ES2322936T3 - MODULATION OF THE T-CELL RESPONSES MEDIATED BY IL-2 AND IL-15. - Google Patents

Abstract

A composition for use as a medicament that includes a first agent that is an interleukin 15 (IL-15) antagonist, wherein the IL-15 antagonist is a mutated IL-15 polypeptide that has at least 65% identity with IL -15, and wherein the IL-15 antagonist binds to and inhibits an activity of IL-15 or an IL-15 receptor, and a second agent that is an interleukin 2 (IL-2) agonist, wherein the agonist is an IL-2 polypeptide, a mutated IL-2 polypeptide or an anti-IL- receptor antibody

Description

Modulation of T cell responses mediated by IL-2 and IL-15.

This request claims the benefit of the U.S. Patent Application No. 60 / 232,251.

Technical field

This invention relates to immunology, transplant rejection, and with diseases associated with the system immune.

Federal government sponsored research

The work described here was supported in part for a grant from the National Institutes of Health. He United States government may, therefore, have certain rights in the invention.

Background

Two of the interleukins, IL-2 and IL-15, are functionally redundant in stimulating the proliferation of T cells in vitro . However, its role in primary immune activation and in vivo immune homeostasis is much less clear. In vivo , IL-2 and IL-15 can have different functions and regulate different aspects of T cell activation. For example, IL-2 can prime apoptosis activated T cells (Lenardo, Nature 353: 858-861, 1991), while IL-15 can support cell survival (Dooms et al., J. Immunol. 161: 2141-2150, 1998; Bulfone et al., Nature Medicine 3: 1124-1128, 1997). IL-15 also appears to direct the proliferation of memory CD8 + T cells in vivo while IL-2 limits its continued expansion (Ku et al., Science 288: 675-678, 2000). In addition, the phenotype of mice deficient in IL-2 is lymphoproliferative and autoimmune (Horak et al., Immunol. Rev. 148: 35-44, 1995), while mice deficient in IL-15 are somewhat lymphogenic and unable to mount a response to the viral challenge (Kennedy et al., J. Exp. Med. 191: 771-780, 2000; Lodolce et al., Immunity 9: 669-676, 1998). The molecular basis for this remarkable dichotomy remains enigmatic.

The functional receptors for IL-2 and IL-15 consist of an isolated α chain, which defines the specificity of the binding for IL-2 or IL-15, and the shared β-receptor of IL-2 and the γ chains. The γ chain is also a critical signaling component of the IL-4, IL-7 and IL-9 receptors (Sugamura et al., Ann: Rev. Immunol. 14: 179-205, 1996). In the lymphoid compartment, these receptor subunits can be expressed individually or in different combinations resulting in the formation of receptors with different affinities and / or with different signaling capabilities (Sugamura et al., See above). For example, the β chain can be associated either with the α chain or with the γ chain to form dimeric structures, or with both chains, the α and β to form trimeric structures. Similarly, the γ chain can interact with the β chain and, through the β chain, with the α chain of either the EL-2 receptor or the E-15 receptor. The α-chain of the IL-15 receptor alone, in contrast to the α-chain of the IL-2 receptor, can be linked to IL-15 with remarkably high affinity (Giri et al., EMBO J. 14: 3654-3663 , nineteen ninety five). However, similar to the α-chain of the IL-2 receptor, it is not believed that this interaction triggers signaling events. Therefore, the trimerization of the chain subunits
α, β and γ is essential for the functional integrity of high affinity receptors for both IL-2 and IL-15.

In vitro studies have shown that activated T cells can express both the α-chain of the IL-2 receptor and the α-chain of the IL-15 receptor (Chae et al., J. Immunol. 157: 2813- 2819, 1996) and the? And? Chains are constitutively expressed by activated T cells (Ishii et al., Int. Immunol. 6: 1273-1277, 1994). In addition, both IL-2 and IL-15 are easily detected during immune activation in vivo (Li et al., J. Immunol. 161: 890-896, 1998). Thus, it is not clear how activated T cells distinguish between IL-2, IL-15, and other cytokines that depend on the γ chain in vivo .

Kim et al. (J Immunol. 1998 January 15; 160 (12): 5742-8) describe an antagonist of IL-15 specific to the generated receiver site by mutation of glutamine residues with terminal C of IL-15 up to aspartic acid and genetically link this mutant IL-15 with the 2α range of Fc from murky These IL-15 mutant proteins are specifically bind to the IL-15R.

Bulfone-Paus and collaborators (Cytokine. 1997 July; 9 (7): 507-13) describe a differential regulation of human T lymphoblast functions by means of IL-2 and IL-15. They they selected the functional differences between IL-2 and IL-15 with respect to control of the functions of the T cell.

WO 97/41232 describes an antagonist polypeptide of IL-15, particularly a polypeptide of IL-15 that has a mutation at position 156 and optionally at position 149 compared to the IL-15 wild type. Additionally, the Polypeptide can be fused to the Fc region of IgG.

Zheng et al. (J Immunol. 1999 October one; 163 (7): 4041-8) describe a protein of IL-2 / Fc cytolytic fusion targeting the receptor IL-2, which can be used to treat Diabetogenic autoimmunity in non-obese diabetic mice.

Summary

The present invention is based, in part, on expression studies of the IL-2 and IL-15 receptor subunits by means of cyclisation of T cells in vivo . Surprisingly, these subunits direct the responses of the activated T cells to IL-2 or IL-15 in a selective manner and, therefore, regulate the response of the T cells in vivo . In other words (and contrary to the conventional wisdom that EL-2 and IL-15 are redundant), IL-2 and IL-15 perform different roles in controlling the proliferation of T cells in vivo . In particular, IL-5 is critical in initiating the division of T cells, while EL-2 controls the expansion of T cells through the reduction of γmac expression. Therefore, in one embodiment, the invention generally characterizes new combinations of IL-2 agonists and IL-15 antagonists and methods for suppressing the immune response through the administration of those agonists and antagonists. In each case, suppression is achieved by administering a first agent that antagonizes an IL-15 molecule or an IL-15 receptor (IL-15R) and a second agent that agonizes an IL- molecule. 2 or an EL-2 receiver (Il-2R). In alternative embodiments, the compositions of the invention may include (instead of, or in addition to, the agents described above), agents that inhibit the expression of nucleic acids (e.g., DNA or RNA) encoding an interleukin (i.e. , IL-15) or an interleukin receptor (that is, the IL-15 receptor).

In a first aspect, the invention relates to with a composition for use as a medicament comprising a first agent that is an interleukin antagonist 15 (IL-15), where the antagonist of IL-15 is a mutated polypeptide of IL-15 that has at least 65% identity with IL-15, and where the antagonist of IL-15 binds to and inhibits an activity of IL-15 or an IL-15 receptor, and a second agent who is an interleukin agonist 2 (IL-2), where the agonist is a polypeptide of IL-2, a mutated polypeptide of IL-2 or a receptor antibody of anti-IL-2.

Another aspect of the invention relates to the use of IL-15 antagonists, where the IL-15 antagonist is a mutated polypeptide of IL-15 that has at least 65% identity with IL-1, and where the antagonist of IL-15 binds to and inhibits an activity of IL-15 or an IL-15 receptor, and a IL-2 agonist, where the agonist is a IL-2 polypeptide, a mutated polypeptide of IL-2, or a receptor antibody anti-IL-2 for the preparation of a therapeutic composition to suppress an immune response in a patient.

A further aspect of the invention is the use of an IL-15 antagonist, wherein the antagonist of IL-15 is a mutated polypeptide of IL-15 that has at least 65% identity with IL-15, and where the antagonist of IL-15 binds to and inhibits an activity of IL-15 or an IL-15 receptor, and a IL-2 agonist where the agonist is a IL-2 polypeptide, a mutated polypeptide of IL-2, or a receptor antibody anti-IL-2 for the preparation of a therapeutic composition for the treatment of a disease malignant IL-15R +.

More generally, the invention offers new combinations of agents that, when administered to a patient, reduce the number of T cells that react with the antigen. For example, the invention offers compositions (for example, pharmaceutically acceptable compositions) that include two or more agents, each of which promotes the death of T cells. Alternatively, the composition may contain at least an agent that promotes the death of T cells and at least one agent which inhibits the proliferation of T cells. The agent that promotes T cell death can promote AICD (induced cell death by activation), passive cell death, ADCC (cytotoxicity antibody dependent cell) or CDC (cytotoxicity dependent  of the complement).

Agents that promote the IACD include the IL-2 molecules and related molecules (by example, IL-2 / Fc or other molecules that work as IL-2 or receptor agonists of IL-2 (for example, an antibody that binds specifically to the IL-2 receptor and mimics the natural ligand binding of the receptor)). Another agent that The IACD promotes is the Fasting Ligand, (FasL). The agents that promote passive cell death include agents that antagonize IL-15 (by selection, by for example, by binding to, and therefore inhibiting activity of, IL-15, an IL-15 receptor, or a component of the intracellular signaling path that is activated once the receiver is linked) or any other factor required for T cell survival (for example, IL-4, IL-7, ligand OX-4, IFN-?, 4-1BB, or IGF-I). In modalities Alternatively, the compositions of the invention may include (in place of, or in addition to, one or more of the agents described above), agents that inhibit the expression of acids nuclei (for example, DNA or RNA) that encode an interleukin (that is, IL-15) or an interleukin receptor (that is, the IL-15 receptor).

ADCC or CDC can be promoted through the exposure of a T cell to an agent that binds to the surface  of the T cell and contains a portion of Fc that activates the ADCC or the CDC More specifically, agents that promote the ADCC or the CDC include fusion proteins that contain a interleukin (for example, IL-2 or a Mutant IL-15) and an Fc region (for example, IL-2 / Fc) as well as antibodies or other proteins that contain Fc that bind to an interleukin receptor (by example, an IL-2 or a receiver of IL-15).

As stated above, the compositions of the invention may include not only a agent that promotes the death of T cells, if not also a agent that inhibits the proliferation of T cells. The agents that inhibit T cell proliferation include rapamycin, mofetil mycophenolate (MMF), azathioprine, and any of the other agents known and used in the art to prevent proliferation cellular (including chemotherapeutic agents). the use of a agent that inhibits T cell proliferation is particularly useful in combination with agents that promote IACD and also stimulate proliferation of T cells (such as IL-2 / Fc). For example, the invention offers a pharmaceutically acceptable composition that includes IL-2 / Fc (which promotes, for example, the IACD and the cell lysis through ADCC or CDC), an antagonist of IL-15 (which promotes, for example, cell death passive through the antagonization of IL-15, a required factor for T cell survival), and rapamycin (which inhibits the proliferation of T cells). They describe later on the compositions containing other combinations of Agents Notably, when two or more agents are employed, no They need to be physically separated from each other. While a agent can be a single entity that has mainly a functional activity (for example, an antibody that directs IL-2 or IL-15 through IL-2 specific binding or IL-15), can also be an individual entity which has at least two functional activities (for example, IL-2 / Fc, mIL-15 / Fc, or an antibody anti-IL-2 or anti-IL-15; in these molecules you are molecules, the interleukin mediates the AICD and the Fc portion of the middle molecule the CDC and the ADCC). Thus, the composition that includes (1) an agent that induces IACD, (2) an agent that induces CDC, and (3) an agent that inhibits cell proliferation can include only two active ingredients (for example, (1) one IL-2 / Fc molecule, which induces AICD and CDC, and (2) rapamycin, which inhibits cell proliferation).

The compositions described here are useful in the treatment of patients who would benefit from the immunosuppression (for example, a patient who has received or is scheduled to receive, a transplant; a patient who has a immune disease, particularly an autoimmune disease; a patient who has cancer (for example, a cancer of a system immune), or a patient suffering from a graft versus disease guest (GVHD)). GVHD is characterized by a response from donor leukocytes against antigens in the receptor. This answer It is particularly problematic in bone marrow transplants, but It also occurs in whole organ transplants; the leukocytes of donors resident in transplanted organs They are always co-planted.

Although the compositions of the invention may contain more than one agent, the methods of the invention are not limited to those in which agents are administered in the form simultaneous. For example, a patient could receive a composition which contains an IL-15 antagonist or a IL-15R antagonist before receiving a composition containing an IL-2 agonist. In similarly, a patient could receive a composition that contains rapamycin before receiving a composition that contains a IL-2 agonist.

In addition, the compositions of the invention (applied simultaneously or sequentially) can be use to treat a cell or organ graft before being Implanted to a patient. The agents of the invention, and the methods for its use, are described further below.

Many of the agents used in the context of the present invention have therapeutic characteristics advantageous For example, agents who run a IL-15R may be fusion proteins that include a an IL-15 mutant polypeptide (mIL-15). These agents are probably not immunogenic because the mutant portion of IL-15 fusion protein may differ from the IL-15 wild type only for a few substituted waste In addition, since the polypeptides of mIL-15 can link to the IL-15Rα with high affinity, they can effectively compete with the IL-15 type wild by the recipient. In addition, the agents of the invention they can activate the host's immune system components such like complement and phagocytes, which ultimately mediate the elimination of (or depletion of) the cells that support the receiver (for example, a receiver of IL-2) to which the agent binds. For example, the agents of the invention can mediate lysis or phagocytosis of target cells Since the receptor alpha subunit of IL-15 (IL-15Rα) is expressed by means of malignant or activated immune cells, but not by resting immune cells, the agents of the invention can be used to specifically direct those cells that have been activated (for example, antigen activated T cells) or that They have become evil. Thus, although T cells they represent a preferred objective for the agents of the invention, the compositions of the invention can also be used to direct other cells involved in the pathogenesis of disorders immunological, such as other cells of the immune system or cells hyperproliferative tissues.

Unless otherwise defined, all technical and scientific terms used here have the same meaning that people usually understand ordinarily trained in the art to which it belongs this invention.

Brief description of the drawings

Fig. 1 is a representation of a sequence of wild-type IL-15 nucleic acid (SEQ ID NO: 1) and the predicted amino acid sequence (SEQ ID NO: 2).

Fig. 2 is a representation of a sequence nucleic acid mutant IL-15 (SEQ ID NO: 3) and the predicted amino acid sequence (SEQ ID NO: 4). The codon of wild type that encodes glutamine at position 149, CAG, and the wild type codon that encodes glutamine in the position 156, CAA, both have been changed to GAC, which encodes aspartate (These positions (149 and 156) correspond to the positions 101 and 108, respectively, in the polypeptide of the Mature IL-15, which lacks a signal sequence in amino acid 48).

Fig. 3A is a series of graphs that describe the expression of the α, β and γmac chains of the IL-2 receptor by dividing the T cells in a host spleen 3 days after injection intravenous (i. v.) of cells labeled with CFSE. The representative data of six experiments.

Fig. 3B is a series of graphs describing the expression of the α, β and γ chains of the IL-2 receptor by dividing the T cells in vitro . The lymphocytes of CFSE-labeled cells were stimulated with anti-CD3 (2 µg / ml) in vitro for three days. Cell division and expression of IL-2 receptor subunits were analyzed by classification of fluorescence activated cells (FACS).

Fig. 3C is a graph describing the expression of selectin L by means of dividing T cells in vivo . Cells were harvested from lymph nodes of host mice three days after intravenous injection of CFSE-labeled cells and colored with PE-anti-CD62 L mAb. The quadrant was programmed based on cells colored with control isotype mAb.

Fig. 3D is a series of graphs describing the differential expression of the IL-2 receptor α chain between T cells that divide in vivo . CFSE labeled cells were stimulated in vivo for three days and the cells were classified into the second cell division. The classified cells were stimulated again in vitro with anti-CD3 and anti-CD28 for three days. Cell division and expression of the α-chain of the IL-2 receptor were analyzed by means of FACS.

Fig. 4A is a series of graphs describing the expression of the α-chain of the IL-15 receptor by dividing T cells in vivo three days after intravenous injection of CFSE-labeled cells. Cells were stained with the IL-15-FLAG fusion protein, followed by staining with biotinylated anti-FLAG mAb and PE-streptavidin. Cell coloration in the absence of IL-15-FLAG was included as a control.

Fig. 4B is a bar graph describing the different responses of in vivo division of T cells to IL-2 and IL-15 in vitro . CFSE-labeled lymphocytes were stimulated in vivo for three days and cell division was analyzed by CFSE profile examination. T cells were classified into the second cell division and cells (1 x 10 4) were cultured in vitro with IL-2 or IL-15 for two days. Cell proliferation was determined by the absorption of 3 H-TdR. The results are presented as the average CPM ± SD of the triplicate trials.

Fig. 4C is a pair of graphs describing the effect of anti-CD25 treatment on the division of T cells in vivo . Host mice were given the anti-CD25 mAb intraperitoneally (ip) at a rate of 1 mg / day for three days immediately before intravenous injection of CFSE labeled cells. Mice treated with the control isotype mAb (rat IgG 1) were included as control.

Fig. 5A is a series of graphs describing the coloration of intracellular IL-2 T cells that divide in vivo . CFSE labeled cells were stimulated in vivo for three days. Cells harvested from the host spleen were stimulated in vitro with PMA and ionomycin for four hours in the presence of GolgiStop ™. The cells were then fixed, permeabilized, and colored for the production of IL-2. Colored cells with the control isotype mAb were included as control.

Fig. 5B is a pair of graphs that describe γ expression by CD4 + T cells of mice efficient in IL-2. The colored ones spleen cells of mice deficient in IL-2 and in wild type control mice with CD4 mAb PE-anti-mouse and mAb of the receptor? IL-2 FITC-anti-mouse. It was analyzed by FACS γ expression by means of CD4 + T cells.

Fig. 5C is a series of graphs describing the effect of anti-CD25 treatment on the expression of γmac by dividing the T cells in vivo . Anti-CD25 mAb host mice were given at a rate of 1 mg / day (ip) for three days immediately before intravenous injection of CFSE-labeled cells. The expression of the β and γ chains of the IL-2 receptor on the division of T cells in vivo was determined on day three (i.e., three days after the injection of CFSE-labeled cells). Mice treated with a control isotype mAb (rat IgG 1) were included as a control.

Fig. 5D is a series of graphs describing the death of apoptotic T cell cells that divide in vivo . CFSE labeled cells were stimulated in vivo for three days. Host spleen cells were harvested and stained with PE-annexin V. Cell division and apoptotic cell death were analyzed by means of FACS.

Fig. 5E is a series of graphs describing the intracellular expression of Bcl-2 by dividing the T cells in vivo . CFSE labeled cells were stimulated in vivo for three days. Cells harvested from the host spleen were colored with Bcl-2 PE-anti-mouse mAb or a control isotype mAb. Cell division and Bcl-2 expression were analyzed by means of FACS.

Fig. 6 is a graphic bar describing the results of an experiment in which cell death (evaluated by means of the release of the 51 Cr isotope from the cells CTLL-2; see the x axis) was evaluated after Treatment with different agents. NP40 is a detergent; IL-2 / Fc is a fusion protein that contains IL-2 and the Fc region of an IgG molecule (this molecule is lithic); C 'is rat complement; IL-2 / Fc - / - is a non-lytic fusion protein that contains IL-2; and mig is an immunoglobulin of murky This study supports the conclusion that IL-2 / Fc cytolytic smooth to cells CTLL-2 that support IL-2R, but IL-2 / Fc non-lytic does not.

Fig. 7 is a series of histograms. It was measured FcRI fluorescence intensity on CHO cells (Chinese hamster ovary) after the cells were exposed to a phosphate buffered saline solution (PBS; part upper left), a murine immunoglobulin (mlgG2a; part upper right), a non-lytic molecule of IL-2 / Fc (IL-2 / Fc - / -; bottom left), and a lytic IL-2 that contains protein of fusion (IL-2 / Fc; lower right). In each histogram, the number of cells is plotted against the intensity of FcRI / CHO fluorescence. This study supports the conclusion of that cytolytic IL-2 / Fc binds to FcRI, but that the non-lytic IL-2 / Fc does not.

Fig. 8 is a series of eight graphs describing the proliferative response of CD4 + (left side) and CD8 + (right side) T cells in vivo . The cells were labeled with CFSE and stimulated in vivo for three days with a lytic molecule (IL-2 / Fc), a cell proliferation agent (rapamycin (Rap)), or the two agents combined. As a negative control, a group of animals was not treated. IL-2 / Fc was analyzed by its CFSE profile. This study supports the conclusion that rapamycin inhibits proliferative signaling of IL-2.

Fig. 9 is a series of four graphs obtained from an experiment in which CFSE-labeled lymphocytes were stimulated in vivo for three days. The expression of the α-chain of IL-2R on the division of T cells by FACS in animals that were not treated (upper left), only rapamycin (Rap; upper right), only IL-2 was evaluated / Fc (bottom left), or a combination of rapamycin and IL-2 / Fc (Rap + IL-2 / Fc; bottom right). This study supports the conclusion that treatment with rapamycin and with IL-2 / Fc promotes the expression of the α-subunit of IL-2R during early proliferation of T cells in vivo .

Fig. 10 is a pair of graphs obtained when CFSE labeled lymphocytes were stimulated in vivo for three days and analyzed by means of FACS. The expression of Annexin V on the division of T cells (CD4 +) in animals that were not treated (left side panel) or rapamycin and IL-2 / Fc (Rap + IL-2 / Fc) was evaluated ; right side panel). This study supports the conclusion that treatment with rapamycin and with IL-2 / Fc promotes apoptosis of CD4 + cells during early proliferation of T cells in vivo .

Fig. 11 is a Table showing the results of the experiments that examined the survival of islet grafts of individuals of the same species in mice non-obese autoimmune diabetics (NOD). Grafts were evaluated in terms of the primary graft function of an individual of the same species (the percentages shown in this column they represent the percentage of mice in which the grafting of an individual of the same species (function was evaluated by monitoring blood glucose levels)) and the average survival time (MST) of the grafts in functioning. n = the number of animals analyzed. The treatments are indicated under the heading "Treatment" (see also the legend that accompanies the Table and the description more down). The results presented here support the conclusion of that treatment with a combination of rapamycin, IL-2 / Fc and mIL-15 / Fc gives as result a lasting survival of islet grafts of individuals of the same species.

Fig. 12 is a Table showing the results of the experiments that examined the survival of skin grafts of individuals of the same species in NOD mice. Grafts were evaluated in terms of the average time of Survival (MST) of the grafts in operation. n = the number of animals analyzed. The treatments are indicated under the "Treatment" heading (see also the accompanying legend Table and description below). The results presented here they support the conclusion that treatment with a combination of rapamycin, IL-2 / Fc and mIL-15 / Fc results in survival lasting of the skin grafts of individuals of the same species.

Fig. 13 is a linear graph that graphs the % of animals that remained free of diabetes during the period of time after treatment with a lytic molecule of EL-2 / Fc, a murine immunoglobulin (mIg), and a non-lytic molecule of IL-2 / Fc. The IL-2 / Fc cytolytic block autoimmunity, but the IL-2 / Fc lithic did not.

Detailed description

An effective immune response begins when an antigen or a mitogen triggers the activation of T cells. In the process of T cell activation, numerous cell changes occur, including the expression of cytokines and cytokine receptors. One of the cytokines involved in the immune response is interleukin 15 (IL-15), which is a growth factor of T cells that stimulates the proliferation and differentiation of B cells, T cells, natural killer cells (NK), and lymphocyte activated killer cells (LAK) in vitro . In vivo , the proliferation of these cell types reinforces the immune response. Another cytokine involved in the immune response, and described here, the IL-2.

The compositions of the present invention include agents that direct IL-15, or its receptor, and to IL-2, or to its receptor, and methods in which those compositions are used to suppress a immune response (for example, a cellular immune response or humoral). Patients benefit from the suppression of immune response in a number of circumstances, for example, in the event of an organ transplant or an immune disease, particularly an autoimmune disease, or Graft Disease versus guest. In other circumstances, for example when Selected immune cells become malignant or self-aggressive, It is convenient to destroy them actively.

The present invention is based on the discovery of new ways to inhibit the immune response. Inhibition can be achieved by administering a combination of agents, one of which targets IL-15 or an IL-15R and one of which targets IL-2 or an IL-2R (forms of administration , including ex vivo graft treatment, are known in the art and are described below). More generally, the number of T cells reactive with the antigen can be reduced by activating signaling pathways that lead to the death of activated T cells (for example, by means of AICD); depriving cells of factors that are required for their survival (cells that die after such deprivation are said to die from passive cell death); or directing the cells activated for lysis by the components of the immune system (cells that die in this form are said to die by means of ADCC or CDC). Therefore, the compositions of the invention include agents that achieve one or more of these purposes (that is, they promote the death of T cells through a recognized cell death pathway (eg, AICD, passive cell death, ADCC, or CDC)). In addition to containing one or more agents that promote T cell death, the compositions of the invention may include one or more agents that inhibit T cell proliferation (as occurs, for example, in response to an antigen). For example, the invention offers a composition (for example, a pharmaceutically acceptable composition or a formulated for application to an organ or cell culture) that includes IL-2 / Fc (for example, which promotes AICD and cell lysis through of ADCC or CDC), mIL-15 / Fc (which antagonizes IL-15 (and therefore promotes passive cell death) and promotes cell lysis through ADCC or CDC), and rapamycin (which inhibits proliferation of T cells).

The term "agent" means that it encompasses essentially any type of molecule that can be used as a therapeutic agent. Proteins, such as antibodies, fusion proteins and soluble ligands, any of which may be either identical to a wild-type protein or contain a mutation (ie, a deletion, an addition, or substitution of one or more residues of amino acids), and the nucleic acid molecules that encode them (or that are "antisense" with them; for example, an oligonucleotide that is antisense with the nucleic acids encoding IL-2, IL-15, or a component ( for example, a subunit) of their receptors), they are all "agents." The agents of the invention can be administered either systemically, locally, or by means of cell-based therapies (that is, an agent of the invention can be administered to a patient by administration of a cell that expresses that agent to the patient). The cell may be a cell administered to the patient solely for the purpose of expressing the therapeutic agent. The cell can also be a cell of a cell, tissue or organ transplant. For example, transplanted cells (for example, islet cells) or cells within tissues or organs (for example, cells within a part of skin, or liver, kidney, or heart) can be treated with an agent or transduced with a nucleic acid molecule that encodes an ex vivo agent (for example, before transplantation). In this way, the transplant cells produce their own immunosuppressive agents. For example, a cell with a desirable phenotype (for example, a cell that produces insulin) can be modified to include a gene that produces one or more of the immunosuppressive factors of the invention. The transplanted cell, tissue, or organ can be treated either before or after the transplant. The methods for administering agents to patients (or to cells or organs in culture) are known and those ordinarily skilled in the art use them routinely and are discussed below.

Agents Directing IL-15 or a IL-15R

Compositions of the invention may include one or more agents that direct IL-15 or a IL-15 receptor. As noted above, A single agent can have multiple functional domains. The agents that direct IL-15 or a IL-15R includes agents that bind to (or well they interact with) IL-15, a IL-15R, or the nucleic acids that encode them like this as agents that bind to and subsequently destroy the cells that support IL-15R, such as T cells activated In this way, the agents useful to achieve the Immunosuppression may contain two functional halves: one objective fraction that directs the agent to a cell that supports an IL-15R and a fraction that depletes T cells objective (eg, lithic) that lead to the elimination of cells that support TL-15R. In one embodiment, the target fraction binds to an IL-15R without effectively transduce a signal through that receiver. By example, the target fraction may include a polypeptide IL-15 mutant, and the fraction that depletes the target T cells can include the Fc region of a molecule of immunoglobulin The Fc region can be derived from an IgG, such as IgG1, IgG2, IgG3, IgG4 or similar mammalian IgG or a IgM, such as human IgM or mammalian analog IgM. In a preferred mode, the Fc region includes the hinge domains, CH2 and CH3 of human IgG1 or murine IgG2a. Although the invention does not is limited to agents that work through any mechanism In particular, it is believed that the Fc region mediates targeted elimination of the complement and phagocyte of the cells that support a IL-15R.

IL-15 mutant polypeptides have been produced that bind the IL-15 receptor complex with an affinity similar to that of wild-type IL-15, but fail to fully activate signal transduction. These mutant polypeptides effectively compete with wild-type IL-15 polypeptides and can inhibit one or more of the events that normally occur in response to IL-15 signaling, such as cell proliferation. The "wild-type IL-15 polypeptide" mentioned herein is a polypeptide that is identical to a naturally occurring IL-15 (for example, a wild-type IL-15 polypeptide is shown in Fig. 1). In contrast, an "IL-15 mutant polypeptide" is a polypeptide that has at least one mutation relative to wild-type IL-15 and that inhibits at least one of the in vivo or in vitro activities that are usually promoted by the IL-15 wild type.

An IL-15 mutant polypeptide which can be used in accordance with the present invention generally block at least 40%, more preferably at least 70%, and most preferably at least 90% of one or more of the activities of the IL-15 type molecule wild. The ability of a mutant polypeptide of IL-15 to block the activity of the IL-15 wild type can be evaluated by of numerous assays, including the cell proliferation assay BAF-BO3 described here (in which the cells were transfected with a construct that encodes a IL-2Rβ). In addition the mutant polypeptides of the invention can be defined according to the percentage of particular identity that they exhibit with the IL-15 wild type. When the percent identity is examined between two polypeptides, the length of the sequences compared it will generally be at least 16 amino acids, preferably at less than 20 amino acids, more preferably at least 25 amino acids, and most preferably at least 35 amino acids. He term "identity", as used in reference to polypeptide or DNA sequences, refers to the identity between subunits (amino acid residues of proteins or nucleotides of DNA molecules) within the two polypeptide sequences or from DNA that are being compared. When the position of a subunit in both molecules it is occupied by the same monomeric subunit (that is, the same amino acid or nucleotide residue), then the molecules are identical in that position. The similarity between two amino acid sequences or two nucleotide sequences is a Direct function of the number of identical positions. In this way, a polypeptide that is 50% identical to a reference polypeptide of 100 amino acids in length can be a polypeptide of 50 amino acids that is completely identical to a portion of 50 amino acids in length of the reference polypeptide. Could be also a 100 amino acid length polypeptide that is 50% identical to the reference polypeptide throughout its length. Since then, many other polypeptides will meet the same criteria. THE identity is typically and more conveniently measured using a software for sequence analysis, such as the Package of Sequence Analysis Software of the Genetics Computer Group of University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, WI 53705), with parameters by defect of it.

An IL-15 mutant polypeptide of the invention has at least 65%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% (for example, 96%, 97%, 98% or 99%) of identity with the IL-15 wild type. The mutation can consist of a change in the number or content of waste from  amino acids. For example, the mutant IL-15 can have a greater or lesser number of amino acid residues than the IL-15 wild type. Alternatively, or additionally, the mutant polypeptide may contain a substitution of one or more amino acid residues that are present in the wild-type IL-15. Polypeptide IL-15 mutant may differ from the IL-15 wild type by addition, deletion, or substitution of a single amino acid residue, for example, a addition deletion or replacement of the residue in position 156. In similarly, the mutant polypeptide may differ from the type wild by an addition, deletion, or substitution of two residues of amino acids, for example, residues at positions 156 and 149. For example, the IL-15 mutant polypeptide may differ from wild-type IL-15 by the replacement of aspartate with glutamine in residues 156 and 149 (as shown in Fig. 2). The mutant polypeptides useful as  target agents, such as wild-type IL-15, recognize and bind to a component of IL-15R. In one embodiment, the IL-15 mutation is in the carboxy terminal cytokine domain, which is believed to bind to IL-2Rγ (the receptor subunit of IL-2). Alternatively, or additionally, the IL-15 mutant polypeptides may include one or more mutations within the binding domain of IL-2Rβ (the β subunit of the receptor of THE 2).

Even in the case that a mutant polypeptide of IL-15 contains a substitution of a residue of amino acid on the other, the substitution may be, but not necessarily, a conservative substitution, which includes a substitution within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; Y phenylalanine, tyrosine.

Instead of using, or in addition to using, a IL-15 target polypeptide (for example a mutant polypeptide of IL-15), the agent Therapeutic can be an antibody. For example, IL-15 can be targeted (that is, specifically bound) with an antibody. Similarly, the IL-15R can be targeted with antibodies that bind to a component of IL-15R (for example, to the IL-15Rα subunit). The methods through of which antibodies can be generated, including Humanized antibodies, against a component of IL-15R are well known in the art. The antibodies should preferably be able to activate the complement and phagocytosis, for example, subclasses IgG3 e Human IgG1 (preferably the latter), or the IgG2a subclass of murky

The methods of the invention can also be carried out with compositions containing: (a) two or more agents, each of which promotes the death of T cells or (b) at least one agent that promotes the death of T cells and at less an agent that inhibits the proliferation of T cells. The agent that promotes the death of T cells can do this by promoting the passive cell death, which occurs when a T cell is deprived of a required factor for its survival. IL-15 is one such agent (others are described later). Thus, agents that interfere with the ability of IL-15 to serve as a factor of survival (for example, an antibody that binds specifically to IL-15 or the receiver of IL-15) can be included in the compositions of the invention (for example, a composition may include an agent that promotes the IACD, an agent that promotes the passive death of cells (for example, an antibody anti-IL-15), and optionally, a agent that inhibits the proliferation of T cells.

As described above, the agents useful to achieve immunosuppression can contain two fractions  functional: an objective fraction that directs the agent to a cell that supports an IL-15R (such as the molecule IL-15 mutant just described) and a fraction of depletion of target cells, for example, that either smooth or leads to the elimination of, the cell that supports IL-15R. In this way, the agent can be a chimeric polypeptide that includes a mutant polypeptide of IL-15 and a heterologous polypeptide such as the region IgG Fc and IgM subclasses of antibodies. The Fc region can include a mutation that inhibits complement fixation and Fc receptor binding, or it may be a depletion of target cells (that is, capable of destroying cells by complement binding or through any other mechanism, such as complement lysis that depends on the antibody).

The Fc region can be isolated from a source of natural occurrence, produced recombinantly, or synthesized (such as any polypeptide offered herein invention). For example, an Fc region can be produced that is homologous to the C-terminal domain of IgG by means of IgG digestion with papain. IgG Fc has a weight molecular approximately 50 kDa. The polypeptides of the invention may include the entire Fc region, or a further portion small that retains the ability to lyse cells. In addition, the Fc fragmented or full length regions may be variants of the wild type molecule. That is, they can contain mutations that may or may not affect the function of the polypeptide.

Reference is made here to the agents that "direct" to an interleukin or an interleukin receiver.  Addressing occurs when an agent is directly linked or indirectly to, or interact with, an interleukin or a interleukin receptor in a way that affects the activity of the interleukin or interleukin receptor. Those ordinarily skilled in the art can evaluate the activity through routine laboratory methods. For example, you can evaluate the strength of the signal transduction or other event biological sequence below that is presented, or that is normally would present, after receptor binding. Activity generated by an agent who directs an interleukin or a receiver interleukin may be, but not necessarily, different from the activity generated when an interleukin of natural occurrence it binds to a naturally occurring interleukin receptor. By example, an agent that directs an IL-2 receptor falls within the scope of the invention even if that agent generates substantially the same activity that would occur having been bound the receptor by natural occurrence IL-2. When an agent generates activity that is substantially the same that, or greater than, the activity generated by a ligand of natural occurrence, the agent can be described as an agonist of the receptor (the agent and the natural ligand being examined under the same conditions). When an agent generates activity that is less than the activity generated by an occurrence ligand naturally, the agent can be described as an antagonist of receptor (if the primary interaction of the agent is with the receptor, for example, mIL-15) or interleukin (if the Primary agent interaction is with interleukin; by example, an anti-IL-15 antibody). Here again, activity levels are assessed by natural occurrence agent and receptor analysis (or ligand) under the same conditions.

The Fc region that may be part of the agents of the invention may be "depletion of cells objective "or" depletion of non-objective cells ". An Fc region that is depleted of non-target cells typically lacks a high affinity binding site of the Fc receptor and a C'1q binding site. The site of high affinity binding of the IgG Fc Fc receptor of Murid includes the Leu residue at position 235 of IgG Fc. From this way, the receptor binding site can be destroyed of murine Fc by means of mutation or suppression of Leu 235. By For example, replacing Glu with Leu 235 inhibits the ability to the Fc region to bind to the high affinity receptor of Fc. He Murder C'1q binding site can be destroyed functionally through mutation or deletion of waste Glu 3118 ', Lys 320 and Lys 322 of IgG. For example, the replacement of Ala residues by Glu 318, Lys 320, and Lys 322 produces an Fc IgG1 unable to direct complement lysis that depends on antibody. In contrast, an IgG depletion Fc region of target cells has a receptor binding site of high affinity of Fc and a C'1q binding site. The site of Fc high affinity receptor binding includes the residue Leu at position 235 of IgG Fc, and the binding site C'1q includes residues Glu 318, Lys 320, and Lys 322 of IgG1. Fc of IgG depletion of target cells has type residues wild or conservative amino acid substitutions in these sites. IgG Fc depletion of target cells can direct to cells for cell cytotoxicity that depends on the antibody or complement-directed cytolysis (CDC). They also know each other appropriate mutations for human IgG (see, for example, Morrison and collaborators, The Immunologist 2: 125, 1994; and Brekke and collaborators, The Immunologist 2: 125, 1994).

The agents who direct the IL-15R can target mutant polypeptides of IL-15, optionally fused to a tag antigenic (for example, a signal sequence). Signal sequences are recognized by anti-FLAG antibodies biotinylated, highly specific, as described here (see also, Blanar et al., Science 256: 1014, 1992; LeClair and collaborators, Proc. Natl Acad. Sci, USA 89: 8145, 1992).

In addition, the α string of IL-15R as an antagonist. Although the complex of IL-15 receptor consists of α subunits β?, the α chain alone shows high affinity by IL-15 and prevents IL-15 from link to an IL-15R complex bound to the cell surface Thus, a soluble α chain of IL-15R can act as an antagonist specific to the receiver.

The construction of the soluble α chain of IL-15R involves fragment cloning extracellular of the α-chain of IL-15R of the positive receptor cells, such as activated T cells or cell lines that express the receptor, and, optionally, fuse it to a sequence of a molecular tag. Sequence of the tag can be, for example, FLAG, GST, or Histidine. This genetic construction in an expression vector can be transfected into expression cell lines. The α string soluble labeled IL-15R produced by lines cell expression will be purified using the mAbs specific to the sequence of the Label. In addition, you can bind an extracellular domain of IL-15R (by example, merged through a peptide link) to a domain Immunoglobulin Fc (for example, hinge domains, CH2 and CH3 of immunoglobulin G), preferably of a subtype of IgG or IgM. Such fusion protein could be expressed in one type of cell. adequate, many of which are known to those ordinarily trained in art.

Agents Directing IL-2 or a Recipient of IL-2

To inhibit an immune response, agents that direct IL-15R-supporting cells, described above, can be administered with an agent that directs IL-2 or IL-2R. An agent that is administered "with" another may be, but not necessarily administered at the same time or in the same way (although this comment is presented in the context of a discussion of agents related to IL-2, it is applicable to anyone of the agents or molecules combined in the compositions of the invention). For example, an agent that directs an IL-15R can be administered before or after an agent that directs an IL-2R. Similarly, an agent that directs IL-15 or an IL-15R (to treat, for example, a cell, a tissue, or an organ exposed to transplantation) can be administered ex vivo while a systemically administered a agent that directs IL-2 or an IL-2R (for example, intravenously) to a patient (for example, a patient who has received a transplant that was treated ex vivo with an agent that directs IL-15). Similarly, an agent that promotes AICD can be administered at a different time or in a different way than an agent that inhibits cell proliferation. Thus, any of the agents or types of molecules that are combined in the compositions of the invention can be administered separately in the methods of the invention.

Agents that direct IL-2 or to an IL-2R include agents that bind to IL-2 or an IL-2R as well as agents that bind to, and subsequently destroy the cells that support an IL-2R, such as T cells activated As described above in the context of the IL-15 address, useful agents to achieve immune suppression may contain a fraction that directs the agent to a cell that supports IL-2R and a depletion fraction of the target cells (for example, lytic) that leads to the elimination of the cell that supports IL-2R. In the event that an Fc region is included, that region can be derived from the same molecules of immunoglobulin described above.

Target agents, such as an agent IL-2 / Fc (see, for example, Zheng et al., J. Immunol 163: 4041-4048, 1999) can be administered with a agent that prevents IL-2R signaling mediated by IL-2, such as rapamycin. The agents that inhibit cell proliferation are well known to those normally trained in art (and discussed further later).

As explained above, the methods of invention (for example, methods to inhibit a response immune (for example, an immune cellular response), the methods for inhibit the rejection of a transplant, and the methods to treat the cancer) can also be carried out with compositions (for example, pharmaceutically acceptable compositions) containing: (a) two or more agents, each of which promotes the death of T cells or (b) at least one agent that promotes cell death T and at least one agent that inhibits the proliferation of T cells. agent that promotes T cell death can do it by promoting AICD (activation induced cell death), and such agent include IL-2 and molecules that function as IL-2 agonists. For example, IL-2 / Fc, the IL-2 mutants that retain the ability to bind and transduce a signal through of the IL-2 receptor, and antibodies that bind specifically and agonize the IL-2 receptor (by example, an antibody that specifically binds to the subunit a of the IL-2 receptor) can be included in the Compositions of the invention. Other agents that promote the IACD include the Fasting Ligand (FasL), which stimulates the death of T cells by activating the transduction cascade of the Fas signal on activated T cells, and mutants biologically active thereof.

Agents that Promote Passive Cell Death

Passive death of T cells occurs when a T cell is deprived of an agent required for survival. In addition to IL-15, factors that include IL-4, IL-7, ligand OX-40, IFN?, 4-IBB and IGF-I are essential (that is, T cells die in the absence of each of these factors; see, for example, Tu et al., J. Immunol. 165: 1331-1336, 2000; Tsuda et al., J. Immunol Meth. 236: 37-51, 2000; Bertolino et al., Int. Immunol. 11 : 1225-1238, 1999; Takahashi et al., J. Immunol 162: 5037-5040, 1999; Pilling et al., Eur. J. Immunol. 29: 1041-1050, 1999; Chu et al., J. Immuno. 162: 1896-1903, 1999; and Weinberg et al., Semin. Immunol. 10: 471-480, 1998). T cells can be deprived of one or more of these factors (IL-15, IL-4, IL-7, etc.), for example, by exposure of the cellulose, in vivo or in culture, to agents that selectively select they bind to one or more of the factors or prevent them from interacting with T cells as they normally would (being the result of deprivation, passive death of cells).

Agents that Promote the ADCC or the CDC

ADCC and CDC can be caused by agents that bind to the surface of T cells and that contain an Fc portion of an immunoglobulin molecule that activates ADCC or CDC. Examples of such agents include antibodies that bind to cell surface structures that are exposed on activated immune cells (eg, cell surface receptors such as CD154, the IL-2 receptor, and the IL-15 receptor). In addition, a chimeric ligand / Fc fusion protein can be used, which binds to the receptor proteins on the surface of activated cells (for example, an IL-2 / Fc or a mIL-15 / Fc). Dices
these examples, other agents suitable for those ordinarily skilled in the art will be apparent.

Agents that Inhibit Cell Proliferation

Agents that inhibit cell proliferation include rapamycin (Sirolimus), mycophenolate mofetil (MMF), azathioprine, and any other agents known to be useful for the treatment of hyperproliferative disorders (such like cancer). The chemotherapeutic compounds well characterized include agents that inhibit metabolism of nucleic acid (such as biosynthesis inhibitors of purine and pyrimidine, RNA synthesis inhibitors, and the DNA binding, DNA modification or agents intercalation). These agents are especially useful when the composition used, for example, to inhibit a response immune, it also contains an agent such as IL-2 / Fc, which not only promotes the IACD but also stimulates the T cell proliferation

Agents that inhibit cell proliferation they also include folic acid antimetabolites such as methotrexate (MTX) and pyrimethamine; purine antimetabolites (such such as 6-mercaptopurine (6-MP) and azathioprine) and pyrimidine antagonists such as cytarabine (ara-C), 5-azacitidine, and 5- fluorouracil (these categories were mentioned above); alkylating agents and other DNA binding agents (for example, cyclophosphamide (CPA); mitomycin C, and Doxorubicin (Adriamycin)); vinca alkaloids (for example vincristine); calcineurin inhibitors (for example, cyclosporin A, FK506, and Brequinar)

Other agents that can be used for inhibit cell proliferation include agents that directly interfere with the proteins involved in the cell cycle regulation (such as anti-CDK (Cell Division Kinase) or anti-cyclines) or proteins that affect the points of cell proliferation check (all the cells that proliferate have checkpoints at different stages of the cycle cell that prevent them from entering the next stage of the cycle of cell division (CDC) before they have completed the previous stage). The routes that feed the point controls of screening include inhibitors of the synthesis of DNA, RNA and protein (for example, S6 kinase inhibitors and PI-3 kinase inhibitors). It can be used also cytokinesis inhibitors.

Procedures for selecting Agents that Inhibit the Response Immune

In addition to analyzing a candidate agent (for example, a mutant IL-15 or an IL-2 polypeptide) in the in vitro assays described in the examples below, any of the following in vitro assays can be used to analyze which particular combinations of the agents described here more effectively achieve immunosuppression. For example, one or more of the agents that target IL-15R can be analyzed in combination with one or more of the agents that antagonize IL-2 or its receptor. These in vivo assays represent only some of the routine ways in which someone ordinarily skilled in the art could further analyze the efficacy of the agents of the invention. They were selected to include them here because of their relevance to the variety of clinical conditions sensitive to treatment with agents that target IL-2, IL-15, and their receptors. For example, the assays are relevant for an organ transplant, an immune disease, particularly an autoimmune disease, a graft versus host disease and cancers of the immune system (for example, cancers that arise when T cells become malignant).

Transplant Paradigms

To determine if a combination of agents the invention achieves immunosuppression, the combination (either directly, through therapy based on genes, or through cell-based therapy) in the context of well established transplant paradigms.

The agents of the invention, the molecules of nucleic acid that encode them (or that hybridize with them and so both inhibit them), can be administered systemically or locally by a standard means for any conventional animal laboratory, such as a rat, a mouse, a rabbit, a rabbit of guinea pigs, or a dog, before carrying out a skin graft of an individual of the same or another species, a transplant of organ, or an implant of cells on the animal. The strains of mice such as C57B1-10, B10.BR, and B10.AKM (Jackson Laboratory, Bar Harbor, ME), which have the same base genetics but do not match in the H-2 locus, are suitable for evaluating different organ grafts.

Heart transplant

A method to carry out cardiac grafts through anastomosis of the donor's heart with the bases older in the abdomen of the host, was first published by Ono and collaborators (J. Thorac. Cardiovasc. Surg. 57: 225, 1969; see also Corry et al., Transplantation 16: 343, 1973). By In the middle of this surgical procedure, the aorta of the donor heart to the abdominal aorta of the host, and it anastomous pulmonary artery of the donor's heart with the vein adjacent cava using standard microvascular techniques. A once the heart is grafted in place and heated to 37 ° C with Ringer's lactate solution, normal sinus rhythm resumes. The function of the transplanted heart can be evaluated frequently by palpation of ventricular contractions to through the abdominal wall. Rejection is defined as cessation of myocardial contractions. The agents of the invention (by example, a combination of mutant IL-15 / Fc, IL-2 / Fc, and rapamycin) would be considered effective in reduction of organ rejection if the guests receiving those agents experience a longer period of acceptance of donor heart graft than guests who were not treated.

Skin graft

The effectiveness of different combinations of The agents of the invention can also be evaluated after a skin graft To carry out skin grafts on a rodent, a donor animal is anesthetized and the thickness removed full of the skin of a part of the tail. The receiving animal is anesthetized also and a graft layer is prepared by removing a patch of affected flank skin. Generally, the patch is approximately 0.5 x 0.5 cm. The skin of the skin is shaped donor to adapt to the graft layer, position it and cover it with gauze, and bandage it. The grafts can be positioned daily starting the sixth day after the operation, and considered rejected when more than half of the epithelium Transplanted seems to be not viable. The agents of the invention (by example, a combination of IL-15 / Fc, IL-2 / Fc, and rapamycin) would be considered effective in reducing rejection of the skin graft if the guests who received those agents experienced a longer period of donor skin graft acceptance that guests do not treated.

A typical example of a grafting experiment of skin, whose result demonstrates the usefulness of a composition containing IL-2 / Fc, mIL-15 / Fc and rapamycin, is described in the Examples (below) and summarized in Fig. 12.

Islet Graft Model of an Individual of the Same Species

Islet cell grafts from individuals of the same DBA / 2J species can be transplanted into rodents, such as B6 AFI mice 6-8 weeks old who became diabetic by means of a single intraperitoneal injection of streptozotocin (225 mg / kg; Sigma Chemical Co., St. Louis, MO). As a control, isogenic grafts of islet cells can be transplanted into diabetic mice. Islet cell transplantation can be carried out following published protocols (for example, see Gotoh et al., Transplantation 42: 387, 1986). In summary, donor pancreas are perfused in situ with type IV collagenase (2 mg / ml; Worthington Biochemical Corp., Freehold, NJ). After a digestion period of 40 minutes at 37 ° C, the islets are isolated on a discontinuous Ficoll gradient. Subsequently, 300-400 islets are transplanted under the renal capsule of each recipient. The graft function of the individual of the same species can be monitored by serial blood glucose measurements (Accu-Check III ™; Boehringer, Mannheim, Germany). The primary function of the graft is defined as a blood glucose level below 11.1 mmol / l on day 3 after transplantation, and transplant rejection is defined as an elevation of blood glucose that exceeds 16, 5 mmol / l (on each of at least 2 successive days) after a period of primary graft function.

Autoimmune Disease Models

Autoimmune disease models provide another means to evaluate combinations of the agents of the invention in vivo . These models are well known to those ordinarily skilled in the art and can be used to determine whether a given combination of agents, which includes, for example, an agent that targets an IL-15R, would be therapeutically useful in the treatment of a disease. specific autoimmune when given directly, through genetic therapy or through cell-based therapies.

The autoimmune diseases that have been modeled in animals include rheumatic diseases, such as rheumatoid arthritis and systemic lupus erythematosus (SLE), diabetes type I, and autoimmune diseases of the thyroid, intestine, and the central nervous system For example, animal models of SLE include MRL mice, BXSB mice, and NZB mice and their F_ {1} hybrids. These animals can be crossed with the purpose of studying particular aspects of the process of rheumatic disease; the progeny of the NZB strain develops severe lupus glomerulonephritis when crossed with NZW mice (Bielschowsky et al., Proc. Univ. Otago Med Sch. 37: 9, 1959; see also Fundamental Immunology, Paul, Ed., Raven Press, New York, NY, 1989). Similarly, a change to lethal nephritis in the progeny of couplings NBZ X SWR (Data and collaborators, Nature 263: 412, 1976). The histological appearance of Renal lesions in SNF1 mice have been well characterized (Eastcott et al., J. Immunol. 131: 2232, 1983; see also Fundamental Immunology, above). Therefore, you can use the general health of the animal as well as the appearance histological renal tissue to determine whether the administration of agents that direct an IL-15R and, for example, directed to the IL-2R, can effectively suppress the immune response in an animal model of SLE.

One of the MRL strains of mice that develops SLE, MRL-lprllpr , also develops a form of arthritis that resembles rheumatoid arthritis in humans (Theofilopoulos et al., Adv. Immunol. 37: 269, 1985). Alternatively, experimental rodent arthritis can be induced by injection of type II collagen in rats (2 mg / ml) mixed 1: 1 in complete Freund's adjuvant (100 µl in total) at the base of the tail. Arthritis develops 2-3 weeks after immunization. The ability of nucleic acid molecules encoding agents of the invention (eg, agents that target IL-15R and agents that target IL-2R or that bind to, and inactivate antigen-activated T cells) to suppress a Immune response can be assessed by directing nucleic acid molecules to T lymphocytes. One way to direct T lymphocytes is as follows. Suspensions of spleen cells 2-3 are prepared after the onset of arthritis and incubated with collagen (100 µg / ml) for 48 hours to induce proliferation of collagen activated T cells. During this time, the cells are transduced with a vector encoding the polypeptide agent of interest. As a control, cultures are made in parallel but not transduced or transduced with an "empty" vector. The cells are then injected intraperitoneally (5 x 10 7 cells / animal). The effectiveness of the treatment is evaluated by following the symptoms of the disease for the next 2 weeks, as described by Chernajovsky et al. (Gene Therapy 2: 731-735, 1995). Fewer symptoms, compared to the control, indicate that the combined agents of the invention, and the nucleic acid molecules that encode them, function as immunosuppressants and are therefore useful in the treatment of an immune disease, particularly an autoimmune disease.

The ability of different combinations of agents to suppress the immune response in the case of diabetes Type I can be analyzed in rat BB strain, which was developed from a commercial colony of Wistar rats in The Bio-Breeding Laboratories is Ottawa. These challenges spontaneously develop antibodies against islet cells and insulin, as it occurs with human type I diabetes. Alternatively, NOD mice can be used (non-diabetic obese) as a model system. A typical example of an experiment in which restores blood sugar levels in mice NOD after an islet transplant from a donor of the same species, described below and summarized in Fig. 11. The animals were treated with a combination of IL-2 / Fc, IL-15 / Fc, and rapamycin. He result was a long-term graft.

Autoimmune thyroid diseases have been moderated in chickens. Obese strain chickens (OS) consistently developed spontaneous autoimmune thyroiditis similar to Hashimoto's disease (Cole et al., Science 160: 1357, 1968). Approximately 15% of these birds produce antibodies to parietal stomach cells, such as in the Human counterpart of autoimmune thyroiditis. Manifestations of the disease in OS chickens, which could be monitored in the course of any treatment regimen, include size Body, fat deposits, serum lipids, sensitivity to cold, and infertility.

They can also be induced experimentally models of an autoimmune disease in the central nervous system (CNS) It can induce an inflammation of the CNS, which leads to paralysis, by means of a single injection of brain tissue or the spine with adjuvant in many different animals of laboratory, including rodents and primates. This model called as experimental allergic encephalomyelitis (EAE) is mediated by T cells. Similarly, myasthenia gravis can occur. experimentally induced by means of a simple injection of acetylcholine receptor with adjuvants (Lennon et al., Ann. N.Y. Acad Sci. 274: 283, 1976).

Autoimmune diseases of the intestine can be modeled in mice "with genetic deactivation" of  IL-2 or IL-10, or in mice that They received enemas containing bovine serum albumin.

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Nucleic Acid Molecules Encoding Agents of the Invention

The polypeptide agents of the invention, including those that are fusion proteins (for example, the mutant IL-2 / Fc and IL-15 / Fc molecules) cannot only be obtained by expression of an acid molecule Nucleic in a suitable eukaryotic or prokaryotic expression system in vitro and subsequent purification of the polypeptide agent, but can also be administered to a patient by means of a suitable genetic therapeutic expression vector encoding a nucleic acid molecule. In addition, a nucleic acid can be introduced into a graft cell before graft transplantation. Thus, the nucleic acid molecules encoding the agents described above are within the scope of the invention. Just as the polypeptides of the invention can be described in terms of their identity with wild-type polypeptides, the nucleic acid molecules that encode them will necessarily have a certain identity with those that encode the corresponding wild-type polypeptides. For example, the nucleic acid molecule encoding an IL-15 mutant polypeptide can be at least 65%, preferably at least 75%, more preferably at least 85%, and most preferably at least 95% (e.g., 96%, 97%, 98%, or 99%) identical to the nucleic acid encoding wild-type IL-15. For nucleic acids, the length of the sequences compared will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 110 nucleotides.

Nucleic acid molecules encoding agents of the invention may contain naturally occurring sequences, or sequences that differ from those of natural occurrence, but, due to the degeneracy of the genetic code, encode the same polypeptide. These nucleic acid molecules may consist of RNA or DNA (for example, genomic DNA, cDNA, or synthetic DNA, such as that produced by phosphoramidite-based synthesis), or combinations or modifications of nucleotides within these types of nucleic acids. In addition, nucleic acid molecules can be double stranded or single stranded (that is, either a sense chain or an anti-
sense).

The nucleic acid molecules of the invention are referred to as "isolated" because they are separated from either the 5 'or the 3' coding sequence whereby they are immediately contiguous in the naturally occurring genome of an organism. Thus, nucleic acid molecules are not limited to sequences encoding polypeptides; Some or all of the non-coding sequences that extend upstream or downstream of a coding sequence may also be included. Those ordinarily trained in the art of molecular biology are familiar with routine procedures for isolating nucleic acid molecules. They can be generated, for example, by treating genomic DNA with restriction endonucleases, or by carrying out the polymerase chain reaction (PCR). In the event that a nucleic acid molecule is a ribonucleic acid (RNA), the molecules can be produced by in vitro transcription.

The isolated nucleic acid molecules of the invention may include fragments that are not found as such in a natural state Thus, the invention encompasses molecules recombinants, such as those in which it is incorporated into a vector a nucleic acid sequence (for example, a sequence encoding a mutant IL-15) (for example, a viral vector or plasmid) or in the genome of a heterologous cell (or the genome of a homologous cell, in a different position than the natural chromosomal location).

As described above, the agents of The invention may be fusion proteins. In addition, or instead of, the heterologous polypeptides described above, a molecule of nucleic acid encoding an agent of the invention can contain sequences that encode a "marker" or a "reporter". Examples of marker or reporter genes include β-lactamase, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), aminoglycoside phosphotransferase (neo r, G418 r), dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK), lacZ (encoding β-galactosidase), and xanthine guanine phosphoribosyltransferase (XGPRT). As with many of the standard procedures associated with the practice of the invention, someone skilled in art will be aware that reagents additional tools, for example, of additional sequences that they can serve the function of a bookmark or a reporter.

The nucleic acid molecules of the invention can be obtained by introducing a mutation in an agent of the invention (for example, a molecule of IL-15 or a molecule of IL-2) obtained from any biological cell, such as the cell of a mammal, or produced by routine methods of cloning In this way, the nucleic acids of the invention can be those of a mouse, rat, guinea pig, cow, sheep, horse, pig, rabbit, monkey, baboon, dog or cat. Preferably, the nucleic acid molecules will be those of a human.

The nucleic acid molecules described previously they can be contained within a vector that is able to direct its expression, for example, in a cell that has been transduced with the vector. Therefore, in addition to the agents of polypeptide, expression vectors contain a molecule of nucleic acid that encodes those agents and cells transfected with those vectors are among the modalities preferred.

Suitable vectors for use in the present invention include T7-based vectors for use in bacteria (see, for example, Rosenberg et al., Gene 56: 125, 1987), the pMSXND expression vector for use in mammalian cells. (Lee and Nathans, J. Bill Chem. 263: 3521, 1988), yeast expression systems, such as Pichia pastoris (e.g., the PICZ family of Invitrogen expression vectors, Carlsbad, CA) and baculovirus derived vectors (for example, the expression vector pBacPAK9 from Clontech, Palo Alto, CA) for use in insect cells. Nucleic acid inserts, which encode the polypeptide of interest in such vectors, can be operably linked to a promoter, which is selected based, for example, on the type of cell in which the expression is intended. For example, the T7 promoter can be used in bacteria, a polyhedrin promoter can be used in insect cells, and a cytomegalovirus or metallothionein promoter can be used in mammalian cells. Also, in the case of higher eukaryotes, cell-specific and tissue-specific promoters are widely available. These promoters are named for their ability to direct the expression of a nucleic acid molecule in a given tissue or cell type within the organism. Someone skilled in the art is very aware of numerous promoters and other regulatory elements that can be used to direct the expression of nucleic acids.

In addition to the sequences that facilitate transcription of the inserted nucleic acid molecule, the vectors may contain origins of replication, and other genes encoding selectable markers. For example, the neomycin resistance gene ( neo r ) imparts resistance to G418 to the cells in which it is expressed, and thus allows phenotypic selection of the transfected cells. Other feasible selectable marker genes that allow phenotypic cell selection include different fluorescent proteins, for example, green fluorescent protein (GFP) and variants thereof. Those skilled in the art can easily determine whether a given regulatory element or selectable marker is suitable for use in a particular experimental context.

The viral vectors that can be used in the invention include, for example, retroviral vectors, adenovirals, and adeno-associated vectors, of herpes virus, of ape virus 40 (SV40) and bovine papillomavirus vectors (see, for example, Gluzman (Ed.), Eukaryotic Viral Vectors, CSH Laboratory Press, Cold Spring Harbor, New York).

Eukaryotic or prokaryotic cells that contain a nucleic acid molecule that encodes an agent of the invention and express the protein encoded in that nucleic acid molecule in vitro are also features of the invention. A cell of the invention is a transfected cell, that is, a cell into which a nucleic acid molecule has been introduced, for example a nucleic acid molecule that encodes an IL-15 mutant polypeptide, by means of techniques of recombinant DNA. The progeny of such cells is also considered within the scope of the invention. The precise components of the expression system are not critical. For example, an IL-15 mutant polypeptide can be produced in a prokaryotic host, such as E. coli bacteria, or in a eukaryotic host, such as an insect cell (e.g., Sf21 cells), or mammalian cells. (for example, COS cells, CHO cells, 293 cells, NIH 3R3 cells, or HeLa cells). These cells are available from many sources, including the American Type Culture Collection (Manassas, VA). In the selection of an expression system, it only matters that the components are compatible with each other. Someone ordinarily skilled in art is capable of making such determination. In addition, if guidance is required in the selection of an expression system, Ausubel et al. (Current Protocols in Molecular Biology, John Wiley and Sons, New York, NY, 1993) and Pouwels et al. (Cloning Vectors: A Laboratory Manual, 1985 Suppl. 1987). Eukaryotic cells containing a nucleic acid molecule that encodes the agent of the invention and expresses the protein encoded in such a nucleic acid molecule in vivo with also features of the invention.

In addition, eukaryotic cells of the invention can be cells that are part of a cell transplant, a tissue or organ transplant. Such transplants may include either primary cells taken from a donor organism or cells that were cultured, modified and / or selected in vitro before transplantation to a recipient organism (eg, eukaryotic cell lines, or progenitor cells). Since, after transplantation in a recipient organism, cell proliferation may occur, the progeny of such a cell is also considered within the scope of the invention. A cell, which is part of a cell, tissue or organ transplant, can be transfected with a nucleic acid that encodes an IL-15 mutant polypeptide, and then be transplanted into the recipient organism, where expression of the polypeptide occurs. IL-15 mutant. In addition, such a cell may contain one or more additional nucleic acid constructs that allow the application of screening procedures, for example of specific cell lineages or cell types before transplantation into a recipient organism.

The expressed polypeptides can be purified from the expression system using procedures routine biomedical, and can be used as agents therapeutic, as described below.

Treatment Sensitive Patients

The compositions of the invention are useful in the inhibition of T cells that are involved, or would be involved, in an immune response (for example, a response immune cell) with an antigen; in the inhibition of other cells involved in the pathogenesis of immunological disorders (by example, monocytes, macrophages, and other cells that have antigens such as dendritic cells, NK cells, and granulocytes), and in the destruction of hyperproliferative cells (as observed, for example, in tissues involved in immunological disorders such as synovial fibroblasts (which affect rheumatoid arthritis), keratinocytes (which are affected in psoriasis), or dermal fibroblasts (which are affected in lupus systemic erythematosus). Given these examples, other types of cells that can be usefully directed will be apparent to those ordinarily trained in art. The cells hyperproliferative can also be cancer cells (by example, malignant T cells).

In this way, you can use the compositions of the invention for treating patients suffering from a immune disease, particularly an autoimmune disease, including, but not limited to the following: (1) a disease rheumatic such as rheumatoid arthritis, lupus erythematosus systemic, Sjögren's syndrome, scleroderma, diseases mixed connective tissue, dermatomyositis, polymyositis, Reiter's syndrome or Behcet's disease, (2) type I diabetes or type II, (3) an autoimmune thyroid disease, such as Hashimoto's thyroiditis or Graves' disease, (4) a disease Autoimmune of the central nervous system, such as sclerosis multiple, myasthenia gravis or encephalomyelitis, (5) a variety of pemphigus such as the vulgar pemphigus, the vegetative pemphigus, the Pemphigus foliaceus, Senear-Usher syndrome, or the Brazilian pemphigus, (6) skin diseases such as psoriasis or neurodermitis, and (7) irritable bowel syndrome (for example, ulcerative colitis or Crohn's disease). They can be used also combinations of agents of the invention to treat the acquired immunodeficiency syndrome (AIDS). In a similar way, the methods by which these agents are administered are they can use to treat a patient who has received a transplant of biological or synthetic material, or a combination of both of them. Such transplants may be organ transplants, of tissue or cell, or synthetic grafts seeded with cells, for example, synthetic vascular grafts seeded with cells vascular In addition, patients suffering from GVHD or patients  who have received a vascular lesion would benefit from this method.

Because the invention encompasses the administration of a form of depletion of the target cells of an agent that targets IL-15R (or an IL-2 receptor, or a combination of IL-15 or IL-15R and IL- 2 or IL-2R), it is possible to selectively kill self-reactive or "transplant-destroying" immune cells without massive destruction of normal T cells. Therefore, the invention offers a method of killing cells that express IL-15R in vivo , which includes activated or self-reactive immune cells or "transplant destroyers" or malignant cells. These methods can be carried out by administering to a patient a combination of agents that includes an agent that directs IL-15R and activates the complement system, smooth to cells by means of an ADCC mechanism, or it kills cells that express the wild-type IL-15 receptor complex. This method can be used to treat patients who have IL-15R + leukemia, lymphoma, or other malignant IL-15R + diseases, such as colon cancer.

Formulations for Use and Administration Routes

The methods of the present invention and the therapeutic compositions used to carry them out they contain "substantially pure" agents. For example, in the event that the agent is a polypeptide, the polypeptide is at minus 60% by weight (dry weight) of the polypeptide of interest, by example a polypeptide that binds and destroys cells that support IL-15R. Preferably, the agents (for example, polypeptides) are at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, of the interest. Purity can be measured by any method appropriate standard, for example, column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

Although agents useful in the methods of the present invention can be obtained from naturally occurring sources, they can also be synthesized or manufactured (for example, agents that bind and destroy cells that support IL-15R can be produced by means of the expression of a recombinant nucleic acid molecule). Polypeptides that are derived from eukaryotic organisms or synthesized in E. coli , or other prokaryotes, and polypeptides that are chemically synthesized will be substantially free of their naturally associated components. In the event that the polypeptide is a chimera, it can be encoded by a hybrid nucleic acid molecule that contains a sequence that encodes all or part of the agent (for example, a sequence that encodes an mutant polypeptide of IL-15 and a sequence encoding an Fc region of IgG). The agents of the invention (for example, polypeptides) can be fused to a hexa-histidine tag to facilitate purification of the bacterially expressed protein, or to a hemagglutinin tag to facilitate purification of protein expressed in eukaryotic cells.

The techniques required to develop the Agents of the invention are routine in the art, and can be carried out without resorting to undue experimentation by Someone trained in art. For example, you can create a mutation consisting of a substitution of one or more of the amino acid residues in IL-15 using the PCR-assisted mutagenesis technique described here for the creation of the IL-15 mutant polypeptide in the which glutamine residues were changed at positions 149 and 156 for aspartic acid residues. The mutations that consist of deletions or additions of amino acid residues (in a IL-15 polypeptide or in any of the others useful polypeptides described herein, for example, polypeptides that inhibit costimulation or that link activated T cells) can be also made with standard recombinant techniques. In therapeutic applications, the agents of the invention can be administer with a physiologically acceptable carrier, such as physiological saline solution The therapeutic compositions of the invention may also contain a carrier or excipient, many of which they are ordinarily known by people trained in art. The excipients that can be used include shock absorbers (e.g. citrate buffer, phosphate buffer, acetate buffer, and buffer bicarbonate), amino acids, urea, alcohols, ascorbic acid, phospholipids, proteins (for example, serum albumin), EDTA, sodium chloride, liposomes, mannitol, sorbitol, and glycerol. The Agents of the invention can be formulated in different ways, from according to the corresponding route administration route. By For example, liquid solutions for ingestion or for injection; gels or powders can be made for ingestion, inhalation, or topical application. The methods to elaborate such formulations are known and can be found, for example, in "Remington's Pharmaceutical Sciences."

Administration routes are also good. known by pharmacologists and doctors and include administration intraperitoneal, intramuscular, subcutaneous, and intravenous. Other Additional routes include intracranial administration (by example, intracisternal or intraventricular), intraorbital, ophthalmic, intracapsular, intraspinal, intraperitoneal, transmucosal, subcutaneous topical, and oral. The routes are expected both intravenous or intraarterial are preferred for administration of agents that direct a recipient of IL-15 The subcutaneous route can be used also frequently since the subcutaneous tissue provides a stable environment for polypeptides, from which they are They can release slowly.

In the case of cell-based therapies (gene therapies), cells / tissues / organs can be transfected either by incubation, infusion or infusion before a transplant with a nucleic acid composition, such that the therapeutic protein is express and subsequently release through transplanted cells / tissues / organs within the recipient organism. Likewise, the cells / tissues / organs could undergo a pretreatment by means of perfusion or simple incubation with the therapeutic protein before the transplant with the purpose of eliminating the immune cells associated with the transplant attached to the donor cells / tissues / organs. (Although this is only a secondary aspect, it probably will not have any clinical relevance). In the case of cell transplants, the cells can be administered either through an implantation procedure or with an injection procedure through a catheter through the wall of a blood vessel. In some cases, the cells can be delivered by release into the vasculature, from which they are subsequently distributed through the bloodstream and / or migrate into the surrounding tissue (this is done in the transplantation of cells from
islet, where islet cells are released into the portal vein and subsequently migrate into the liver tissue).

It is well known in the medical art that the doses for any patient depend on many factors, including the general state of health, sex, weight, body surface area and age of the patient, as well as the particular compound that is It will administer, time and route of administration, and other drugs that are administered at the same time. The doses for the polypeptide of the invention will vary, but may, when administered intravenously, be delivered in doses of the order of 1 microgram up to 10 mg / kg body weight or of an order of magnitude of 0.01 mg / l up to 100 mg / l blood volume. One dose may be administered one or more times per day, if necessary, and treatment may be continued for prolonged periods of time. Dose determination
Correct for a given application is within the abilities of the person ordinarily skilled in the art.

Examples Reagents

The following reagents were used in the studies described here: human IL-2 was obtained Hoffman-La Roche recombinant (Nutley, NJ); be obtained rapamycin from Wyeth-Ayerst (Princeton, NJ); cyclosporin-A (CsA) was obtained from Sandoz (East Hanover, NJ); RPMI-1640 and serum were obtained calf fetal (FCS) from BioWittaker (Walkersville, MD); be they obtained penicillin, streptomycin, G418, and strepavidin-RED670 from Gibco-BRL (Gaithersburg, MD); dexamethasone, PHA, lysozyme were obtained, Nonidet P-40, NaCl, HEPES, and PMSF from Sigma (St. Louis, MO); Ficoll-Hypaque was obtained from Pharmacia Biotech (Uppsala, Sweden); IL-15 were obtained recombinant human and anti-human Ab IL-15 of PeproTech (Rocky Hill, NJ); were obtained Ab anti-signal and affinity granules anti-FLAG of International Biotechnologies, Inc. (Kodak, New Haven, CT); pRcCMV was obtained from In Vitrogen Corporation (San Diego, CA); genistein was obtained from ICN Biomedicals (Irvine, AC); Disuccinimidil suberate (DSS) was obtained from Pierce (Rockford, IL); New England restriction endonucleases were obtained Biolabs (Beverly, MA); New England [3 H] TdR was obtained Nuclear (Boston, MA); and conjugated antibodies were obtained with fluorescent dye CD25-PE3, CD 14-PE, CD16-PE, CD122-PE, CD4-FITC, CD8-FITC, IgG1-PE or IgG1-FITC of Beckton / Dickinson (San José, CA). Be synthesized the signal peptide in the Synthesis Facilities of Peptides at Harvard Medical School.

Fusion Protein Production FLAG-HMK-IL-15

To study the cellular pattern of expression of human IL-15 receptor, a plasmid was constructed that could be used to express a fusion protein of IL-15 The plasmid encodes a polypeptide of IL-15 that has an N-terminal covalently linked to the FLAG-HMK sequence of 18 amino acids (FLAG-HMK-IL-15). FLAG sequences are recognized by antibodies anti-FLAG (Blanar et al., Science 256: 1014, 1992); LeClair et al., Proc. Natl Acad Sci. USA 89: 8145,1992) while the HMK sequences (site of Cardiac Muscle Kinase recognition) allow the introduction of the radioactive label [32 P] within the molecule (Blanar et al., see above, LeClair and collaborators, see above).

For the construction of the FLAG-HMK-IL-15 plasmid, a 322 bp cDNA fragment encoding the mature IL-15 protein was amplified by PCR using synthetic oligonucleotides [5'-G GAATTC AACTGGGTG
AATGTAATA-3 'direction (SEQ ID NO: 5; EcoRI site (underlined) plus bases 145-162); 5'-CGGGATCCTCAA
GAAGTGTTGATGAA-3 '(SEQ ID NO: 5 antisense; BamHI site [underlined] plus bases 472-489)]. The DNA template was obtained from human PBMCs activated with PHA. The PCR product was purified, digested with EcoRI and BamHI, and cloned into the plasmid pAR (DRI) 59/60 digested with EcoRI-BamHI as described (Blanar et al., Science 256: 1014, 1992; LeClair and collaborators, Proc. Natl. Acad Sci. USA 89: 8145, 1992). The backbone of plasmid pAR (DRI) 59/60 contains in the frame sequences encoding the FLAG peptide recognition sequences and HMK (Blanar et al., Science 256: 1014, 1992; LeClair et al., Proc. Natl. Acad. Sci. USA 89: 8145, 1992).

Fusion Protein Expression and Purification FLAG-HMK-IL-15

The IL-15 related fusion construct, FLAG-HMK-IL-15, was expressed in E. coli strain BL-21 and purified by affinity with anti-FLAG coated granules as described (Blanar et al. , Science 256: 1014, 1992; LeClair et al., Proc. Natl. Acad Sci. USA 89: 8145, 1992). The affinity column fusion protein was eluted after extensive washing with 0.1 M glycine (pH 3.0). The eluate containing FLAG-HMK-IL-15 was dialyzed against a buffer containing 50 mM Tris (pH 7.4) and 0.1 M NaCl for 18 hours at 4 ° C, filtered through a 0.2 membrane um, and stored at -20 ° C.

FLAG-HMK-IL-15 binds to the IL-15Rα subunit

The purified fusion protein was analyzed FLAG-HMK-IL-15 for determine if it interacts with the receptors of IL-15 of the cell surface. How I know described above, [32 P] - was added FLAG-HMK-IL-15 a PBMC cultures that were activated by a mitogen, PHA With the purpose of permanently binding proteins interactive with each other, the chemical interlacer was added Disuccinimidil suberate (DSS). They were washed, lysed, and centrifuged the cells, and detergent soluble proteins were separated by SDS-PAGE medium. The autoradiography of proteins separated by SDS-PAGE revealed a band single 75-80 kDa corresponding to the combined molecular weight of FLAG-HMK-IL-15 (15 kDa) and the human IL-15Rα subunit (60- 65 kDa). The identity of this band was confirmed as the subunit of IL-15Rα by means of experiments of entanglement performed in the presence of a molar excess of hIL-15. Under these conditions, we fail to detect the 15 kDa band marked radioactively. In this way, the conformation of the fusion protein [32 P] - FLAG-HMK-IL-15 allows specific binding on the subunit site of 60-65 kDa IL-15Rα expressed on the PBMC surface activated by mitogen.

FLAG-HMK-IL-15 It is a Biologically Active Growth Factor that Requires the IL-2R? Expression

In the following series of experiments, analyzed the fusion protein FLAG-HMK-IL-15 for determine if it could work as a growth factor biologically active Human PBMCs activated with PHA proliferate  in response to either FLAG-HMK-IL-15 or a human recombinant IL-2, as detected at through the incorporation test of [<3> H] -TdR. A FLAG peptide that lacks the IL-15 sequence does not stimulate proliferation mobile. As IL-2 does, the fusion protein FLAG-HMK-IL-15 stimulates proliferation of transfectant cells IL-2Rγ + BAF-BO3 which express the IL-2R? subunit. Protein fusion FLAG-HMK-IL-15, without However, it does not stimulate the proliferation of parental cells BAF-BO3 that were transfected with a vector that lacks the sequences of the chain of IL-2Rβ. Thus, FLAG-HMK-IL-15 is a biologically active growth factor that requires the IL-2R? chain expression on the target cells with the purpose of stimulating proliferation mobile.

PBMC Activated by Mitogen but not Remaining, Express to the IL-15Rα Subunit

Fusion protein were used FLAG-HMK-IL-15, the biotinylated anti-FLAG antibody, and streptavidin-RED670 to detect the expression of IL-15 binding sites on PBMCs human by means of cytofluorometric analysis. PBMC analyzed were either freshly isolated or activated by PHA These cells were washed and incubated with either medium alone or with FLAG-HMK-IL-15 followed by biotinylated Ab-FLAG Ab and Streptavidin-RED670. The cells were analyzed colored by flow cytometry. The PBMC that were activated with PHA expressed to the proteins of IL-15R? But not to the remaining PBMCs. In in line with the results of the experiments of entanglement described above, the binding is blocked from FLAG-HMK-IL-15 with PBMC activated by PHA by means of a molar excess of RIL-15, thus demonstrating the specificity of the binding of FLAG-HMK-IL-15 by IL-15 binding sites. Both activated CD4 + cells such as CD8 + express the chains of IL-15α. Activation induced the IL-15Rα chains were also detected on CD14 + cells (monocyte / macrophage) and on cells CD16 + (natural killers).

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The IL-2Rα e Subunits IL-2R? Are not Required for Binding from IL-15

FACS analysis of PBMCs activated by PHA protein colored FLAG-HMK-IL-15 and Mab anti-CD25, against the subunit of IL-2R? Reveal populations of cells that express both the IL-15Rα subunits such as IL-2Rα, as well as cell populations that express any subunit, but not both. exist IL-2Rα + cells that do not bind to FLAG-HMK-IL-15. Almost all PBMCs that were stimulated with PHA only during a day express both chains of IL-15R? as to those of IL-2R? (double positive) and a much smaller population of IL-15R? +. IL-2R? - cells were observed (positive simple). In a very interesting way, there are cells IL-2R? + That fail to bind to IL-15 Therefore, the expression of the chains of IL-2R? Is not sufficient for binding of IL-15.

Taken together, these data indicate that IL-15 can bind to cells IL-15Rα +, IL-2R? -, e IL-2R? -. A conclusion was reached similar through experimentation that proved that the interaction  of IL-15 with cells IL-2R? -,? - transfected with subunits of IL-15R? (Anderson and collaborators, J. Biol. Chem. 270: 29862, 1995; Giri and collaborators, EMBO J. 14: 3654, 1995). In addition to the requirement for the expression of the IL-15Rα subunit, IL-2R? e subunits are required IL-2Rγ to return to cells sensitive to IL-15 activated growth.

In summary, the experiments presented They have previously shown that: (i) the subunits of IL-15Rα are rapidly expressed by means of activated macrophages, T cells, and NK cells, and (ii) induction of the IL-15R? subunit is blocked by dexamethasone medium but not by CsA or rapamycin. In addition, experiments have confirmed that the subunit of IL-15R? Is necessary and sufficient for the binding of IL-15 and that fusion protein FLAG-HMK-IL-15 is an extremely useful tool for studying IL-15 receptors.

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The IL-2R? Subunit is Critical both for IL-2 Signal Transduction as of IL-15

Decreased viability of T cells activated and therefore depletion of activated T cells provides a way to decrease lymphokine production and of mitogens that contribute to accelerated atherosclerosis, a graft rejection of an individual of the same species, certain leukemias and other immune mediated pathologies. In addition, the blocking of the signal transduction path activated by IL-15 also provides a way to decrease the production of lymphokines and mitogens that contributes to a accelerated atherosclerosis, a graft rejection of an individual from the same species, certain leukemias and other pathologies mediated in immune form When activated, T cells proliferate and express receptors on their cell surface to interleukins In addition, activated T cells release at least 3 lymphokines: interferon gamma, factor II differentiation of B cells, and IL-3. These lymphokines can produce different undesirable events, such as graft rejection of an individual of the same species. In contrast, the rest of the T cells and long-term memory of T cells do not express lymphokine receptors This difference in the expression of receptor provides a means to direct the cells immunoactivated without interfering with the rest of the cells. The molecules designed to recognize some subunits of IL-15R will recognize activated monocytes / macrophages as well as activated T cells and can be used to inhibit or Selectively destroy these cells. The derivatives of IL-15 that bind to a subunit of IL-15R but lacking the activity of IL-15, either because they block the binding and / or the Authentic IL-15 absorption, are useful in the method of the invention The mutant IL-15 molecule described below provides a working example of such derivative.

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An IL-15 mutant polypeptide that directs a IL-15R Genetic Construction of IL-15 Mutant

The human IL-15 protein that supports a double mutation (Q149D; Q156D) was designed to direct the putative sites critical for binding to the IL-2Rγ subunit. Polar but not charged glutamine residues at positions 149 and 156 were mutated in acidic aspartic acid residues using PCR-assisted mutagenesis. A cDNA encoding the double mutant of IL-15 was amplified by PCR using a synthetic oligonucleotide sense [5'-GGAATTCAACTGGGTGAATGTAATA-3 '(SEQ ID NO: _); EcoRI site (underlined hexamer) plus bases 145-162 ] and a synthetic antisense oligonucleotide (5'-CG GGATCC TCAAGAA GTGTTGATGAACA TG T C GACAAT-ATGTACAAAACT G T C CAAAAAT-3 '(SEQ ID NO: _); BamHI site ( hexamer underlined) plus bases 438-489; mutated bases are underlined individually.] The template was a plasmid containing cDNA encoding human FLAG-HMK-IL-15. The amplified fragment was digested with EcoRI / BamHI and cloned within plasmid pAR (DRI) 59/60 digested with EcoRI / BamRI as described (LeClair et al., Proc. Natl. Acad Sci. USA 89: 8145, 1989). The presence of a mutation in residue 156 was confirmed by Digestion medium with SalI ; the mutation introduces a new SalI restriction site.In addition, the mutations were verified by DNA sequencing, according to standard techniques.The double mutant protein FLAG-1-nVlK-IL-15 was produced (Q149D; Q156D), it was purified, and verification was done by r sequencing medium as described above for wild-type FLAG-HMK-IL-15 protein.

Using this same strategy, they prepared the mutants that contained a single amino acid substitution, since either in position 149 or in position 156. As described previously, these positions (149 and 156) correspond to the positions 101 and 108, respectively, in the mature polypeptide of IL-15, which lacks a signal sequence of 48 amino acids.

Similarly, you can use this strategy to incorporate any other amino acid instead of glutamine residues in positions 149 or 156 or for introduce amino acid substitutions at positions other than 149 and / or 156.

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Proliferation of BAF-BO3 cells in the presence of IL-15 Related Proteins

The double mutant polypeptide of IL-15 can inhibit the proliferation of BAF-BO3 in a dose-dependent manner: the addition of 30 µL (approximately 50 µg / ml) of Il-15 double mutant inhibited proliferation more completely that the addition made of 20 µL of it concentration of the double mutant IL-15.

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Proliferation of Human PBMC Stimulated with PHA

The ability of the double mutant polypeptide of FLAG-HMK-IL-15 for linking to human PBMCs activated with PHA was demonstrated from the Following way. The activated PBMCs were washed with PHA and were incubation with medium alone, or with the double mutant of FLAG-HMK-IL-15. Be then incubated the cells with a biotinylated antibody anti-FLAG and colored with conjugated streptavidin with RED670. The colored cells were analyzed by means of flow cytometry.

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FACS Analysis of Colored Leukemic Cell Lines with FLAG-HMK-IL-15 of Wild type

In a series of experiments similar to those above, the ability of the polypeptide was shown FLAG-HMK-IL-15 of wild type to bind leukemia cells. The cells treated were obtained from leukemic cell lines MOLT-14, YT, HuT-102, and from cell lines that are currently being established in the Beth Israel Hospital (Boston, MA), and calls 2A and 2B. They washed cultured cells and incubated with either medium alone or with medium containing the polypeptide FLAG-HMK-IL-15 of wild type. The cells were then incubated with the antibody biotinylated anti-FLAG and colored with streptavidin conjugated with RED670. The cells were analyzed colored by flow cytometry.

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Genetic Construction of Mutant Chimeric Polypeptides Additional IL-15

In addition to the chimera of FLAG-HMK-IL-15, which provides the mutant IL-15 with a label antigenic, numerous other polypeptides can be linked to any mutant of IL-15 or IL-2. For example, IL-15 or IL-2 mutants can be linked to the Fc fragment of the IgG subclass of antibodies and according to the following method.

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Genetic Construction of IL-15 / Fc Mutant

The cDNA for Fcγ2a can be generated at from RNA extracted from a hybridoma that secretes IgG2a using  standard techniques with reverse transcriptase (MMLV-RT; Gibco-BRL, Grand Island, NY) and a synthetic oligo-dT oligonucleotide (12 - 18) (Gibco BRL). The mutant IL-15 cDNA can be amplify from a plasmid template by PCR using specific synthetic oligonucleotides of IL-15

The 5 'oligonucleotide is designed to insert a single 40' NotI restriction site 5 'to the transcriptional initiation codon, while the 3' oligonucleotide removes the termination codon and modifies the use of the Ser C-terminal residue codon from AGC up to TCG to accommodate the creation of a single BamHI site at the junction of mutant IL-15 / Fc. The synthetic oligonucleotides used for the amplification of the cDNA of the Fcγ2a domain change the first codon of the Glu hinge by ASP in order to create a single BamHI site that encompasses the first codon of the hinge and introduces a single XbaI site 3 'to the termination codon.

The Fc fragment can be modified so that the target cells are not depleted, that is, they are not able to activate the complement system. To make the construction of the mutant IL-15 not deplete the target cells (mIL-15 / Fc), site-directed oligonucleotide mutagenesis is used to replace the C'lq binding motif of Glu318, Lys320, Lys322 with Ala residues . Similarly, Leu235 is replaced with Glu to inactivate the FcγR binding site I. Cytokine and Fc component ligation in the correct translation reading frame at the single BamHI site produces an open reading frame of 1236 base pairs encoding a single 411 amino acid polypeptide (including the 18-amino acid IL-15 signal peptide) with a total of 13 cysteine residues. It is predicted that secreted mature homodimeric IL-15 / Fc has a total of up to eight intramolecular bonds and three heavy chain intermolecular disulfide bonds and an approximately 85 kDa molecular weight, exclusive of glycosylation.

Expression and Purification of Fc Fusion Proteins from MIL-15 receiver

Proper genetic construction of mIL-15 / Fc can be confirmed by DNA sequence analysis after cloning of the fusion gene as a NotI-XbaI cassette within the eukaryotic expression plasmid pRc / CMV (Invitrogen, San Diego , CA). That plasmid carries a CMV promoter / enhancer, a bovine growth hormone polyadenylation signal and a neomycin resistance gene for selection with G418. Plasmids carrying the mIL-15 / Fc fusion gene are transfected into Chinese hamster ovary cells (CHO-K1, available with the American Type Culture Collection) by electroporation (1.5 kV / 3 µF / 0.4 cm / PBS) and is selected in serum-free Ultra-CHO medium (BioWhittaker Inc, Walkerville, MD) containing 1.5 mg / ml of G418 (Geneticin, Gibco BRL). After subcloning, clones that produce high levels of the fusion protein are selected by selecting IL-15 supernatants through ELISA (PharMingen, San Diego, CA). The mIL-15 / Fc fusion proteins are purified from the culture supernatants by sepharose affinity chromatography with protein A followed by dialysis against PBS and sterilization through a 0.22 µm filter. Purified proteins can be stored at -20 ° C before use.

Western blot analysis followed by SDS-PAGE under reducing conditions (with DTT) and Non-reducing (without DTT) can be carried out using primary anti-Fcγ antibodies or monoclonal anti-mIL-15 or polyclonal to assess the isotype size and specificity of fusion proteins. The functional activity of IL-15 / Fc mutant can be evaluated by means of a standard T cell proliferation assay, as described previously. The following mAbs were obtained from PharMingen (San Diego, CA): CD25 PE-anti-mouse (α-chain of IL-2R, IgG1, PC61), CD122 rat anti-mouse (β chain IL-2R, IgG2b, TM-b1), CD132 rat anti-mouse (IL-2R?, IgG2b, TUGm2), Hamster anti-mouse CD3 (IgG, 145 - 2C11), CD28 hamster mouse (IgG, 37.51), CD62L PE-anti-mouse (IgG2a, MEL14), Bcl-2 anti-mouse hamster conjugated to PE (IgG, 3FI1), IL-2 PE conjugated anti-mouse (IgG2b, JES6-5H4), PE-annexin V, IgG2b biotinylated anti-rat, PE-streptavidin, PE-Cichrome, and mAb control isotype conjugated to PE. A biotinylated mAb mouse anti-FLAG and an IgG1 control mAb from Rat were obtained through Sigma Chemical Co. (St Louis, MO).  An AB cell hybridoma was obtained that secretes the CD25 mAb rat anti-mouse (TIB 222, IgGI) through American Type Culture Collection (ATCC; Manassas, VA). They were grown cells in serum-free UltraCulture culture medium (BioWhittaker, Walkerville, MD) and the mAb was purified in the culture supernatant with a G protein column.

Expression studies of EL-2 and IL-5 in vivo

IL-2 e were acquired Recombinant human IL-15 with R&D System (Minneapolis, MN). The structures were constructed, expressed and analyzed. IL-15-FLAG e fusion proteins Mutant IL-15 / Fc as previously reported (Chae and collaborators, J. Immunol. 157: 2813-2819, 1996; Kim and collaborators, J. Immunol. 161: 5742-5748, 1998). They were used rat anti-mouse mAb gc (4G3 / 3E12, IgG2b) as was previously reported (Li et al. J. Immunol. 164: 1193 - 1199, 2000). The lymphocytes were labeled with the fluorochrome 5-carboxyfluorescein succinimidyl ester diacetate (CFSE; Molecular Probes, Inc., Portland, OR) as follows. Spleens and peripheral lymph nodes of mice were collected donors and individual cell suspensions were prepared in Hanks balanced salt solution (HBSS). Blood cells were lysed red through hypotonic shock. The cells were resuspended in HBSS at a rate of 1x10 7 / ml and marked with CFSE as describe Wells et al. (J. Clin. Invest. 100: 3173-3133, 1997).

To activate CFSE labeled T cells in vivo , 2 mice / DBA (1000 rad) were irradiated with a Gammacell Exactor (Kanata, Ontario, Canada). Each mouse then received 4 to 6 x 10 7 CFSE labeled cells in 0.5 ml of HBSS through the tail vein. Three days later, host mice were sacrificed and peripheral lymph nodes and spleens were collected separately. Individual cell suspensions were prepared for cell surface coloration and FACS analysis.

In some experiments, irradiated host mice were treated with anti-CD25 mAb or anti-gammac mAbs (intraperitoneally, at a rate of 1 mg / day for 3 days with an intravenous injection of CFSE labeled cells). Cell division was determined in vivo on the third day after injection of CFSE labeled cells. Treatment with mutant IL-15 / Fc fusion protein consisted of 1.5 µg daily intraperitoneally, for three days, beginning with an intravenous injection of labeled cells.

CFSE labeled cells activated in vivo in irradiated allogeneic hosts were colored for the expression of IL-2 and IL-15 receptor subunits. To detect the expression of the α-chain of the IL-2 receptor, cells (2 x 10 6) were colored with CD25 PE-anti-mouse mAb on ice for 30 minutes, washed, and resuspended in 1 ml of PBS containing 0.5% BSA. To detect the expression of [gamma] and [beta] IL-2R, the cells were incubated with a rat β-mouse anti-mouse chain (IgG2b) or with mAb [gammac] (IgG2b) on ice for 30 minutes, followed by incubation with an IgG2b biotinylated anti-rat. The cells were washed and further colored with PE-streptavidin for 20 minutes. The cells were washed and resuspended in PBS-0.5% BSA for analysis. To detect expression of the IL-15R α chain, cells were incubated with IL-15-FLAG fusion protein that binds to the α chain (Chae et al., J. Immunol. 157: 2813-2819 , 1996) and then were colored with biotinylated mouse anti-FLAG mAb. The cells were then washed and colored with PE-streptavidin. Control isotype mAbs that match each experiment as control were included. All samples were analyzed using FACSort with the CellQuest? Software (Becton Dickinson, Mountain View, CA). Data were collected and analyzed by means of periodic activation on CFSE + cells. All CFSE + dividing cells were T cells, as defined by CD3 expression. At least 100,000 events were collected for each sample.

Apoptosis of dividing T cells in vivo was analyzed as follows. CFSE-labeled lymphocytes were stimulated in irradiated allogeneic hosts as described above. Cells from the host spleen or peripheral lymph nodes were collected three days later and stained with annexin V conjugated with PE on ice for 15 minutes in labeling buffer. Cell division was identified based on the CFSE profile of the cells, and apoptotic death of the cells in each different cell division was analyzed by annexin V staining.

Cells were also colored for intracellular expression of IL-2 and Bcl-2 cytokines. CFSE-labeled cells that had been activated in vivo for three days from the host spleen and lymph nodes were collected. The cells were stimulated again in vitro with PMA (50 ng / ml) and ionomycin (500 ng / ml) for four hours and GolgiStop ™ (PharMingen) was added during the last two hours of the culture. The cells were fixed and permeabilized with Cytofix / Cytoperm (PharMingen) at 4 ° C for 10 minutes, and then colored with a PE-conjugated anti-mouse IL-2 mAb, the control isotype matching mAb was included as a control . For staining with Bcl-2 the cells were fixed and permeabilized with Cytofix / Cytoperm for 10 minutes and stained with anti-Bcl-2 mAb conjugated to PE or with the Ab isotype control for 30 minutes. The cells were washed and analyzed by means of FACS.

The classification of the cells and the new in vitro stimulation was carried out as follows. CFSE labeled cells were prepared from irradiated allogeneic host three days after intravenous injection of labeled cells. In vivo cell proliferation was identified through the analysis of their CFSE profiles. The second activated cell divisions were selected and classified with the Ventage? For FACS (Becton Dickinson) classifier at a rate of 2,000 events / second. The classified cells were resuspended in RPMI 1640 medium supplemented with 10% FCS and 1% penicillin and streptomycin at a rate of 5 x 10 5 / ml and plated on plates coated with anti-CD3 (2 \ mug / ml) together with the anti-CD28 mAb (1 µg / ml). Three days later, the cells were harvested and stained with anti-mouse CD25 conjugated to PE and Ab isotype control. Cell proliferation and expression of the α-chain of the IL-2 receptor were analyzed by means of FACS. Cell classification and in vitro proliferation assays were performed as follows. CFSE labeled cells were prepared from irradiated allogeneic hosts three days after intravenous injection of labeled cells, and in vivo cell proliferation was identified through analysis of the CFSE profile of the cells. The second activated cell division was selected and classified with Ventage? For FACS. The cells (1 x 10 4 / ml) were resuspended in RPMI 1640 medium with 10% FCS and 1% penicillin and streptomycin, and stimulated with IL-2 (40 µ / ml up to 500 µ / ml) or IL-15 (5 ng / ml) for 48 hours. Cells were pulsed with 1 mCi 3 H-TdR (Amersham, Boston, MA) for 16 hours and assimilation of 3 H-TdR was determined by scintillation counting (Beckman Instruments, Columbia, MD ).

The reagents and techniques described above provided the basis for different findings. First, CFSE-labeled allogeneic B6AF1 lymphocytes (H-2b / dk), in contrast to isogenic controls (Li et al., Nature Medicine 5: 1298-1302, 1999), proliferated vigorously in irradiated DBA / 2 hosts (H- 2d). Approximately 20% of CFSE-labeled T cells recovered from the host spleen entered the cell cycle within three days of the adoptive transfer, and seven to eight discrete cycles of cell division were clearly identified (Fig. 3A). Surprisingly, the α-chain of the IL-2 receptor, which is required for high affinity signaling of the IL-2 receptor, could not be detected during the first 5 divisions, that is, this subunit of the receptor is expressed only after of five cell divisions. In contrast, the β-subunits of the IL-2 receptor were constitutively expressed for all dividing T cells, and their level of expression was progressively increased as the cells continued to divide. The expression pattern of γ in vivo differs strongly from that of the α chain and the β chain (Fig. 3A). Undivided T cells (division 0) expressed very low levels of the γ chain (<10%). After entering the cell cycle, the α chain was highly expressed by dividing T cells, and the increase in expression levels continued after each consecutive cell division. After five cell divisions, however, the γ chain expression was drastically reduced, close to reaching the baseline level after the sixth cell division (Fig. 3A).

The differential expression of the subunits of the IL-2 receptor is not due to accumulation selective of a subset of activated T cells in the spleen host, since CFSE-labeled cells collected at from the peripheral lymph nodes showed a pattern remarkably similar expression for the three subunits of the EL-2 receptor.

Second, stimulation of CFSE labeled T cells in vitro resulted in uniform expression of all three subunits of the IL-2 receptor (Fig. 3B). This suggests that the regulation of IL-2 receptor expression in vivo is different from that in vitro . It is known that the α-chain of the IL-2 receptor is sensitive to proteolytic cleavage in vivo in a manner similar to that of selectins (Hemar et al., J. Cell. Biol. 129: 55-64, 1995). Staining for L-selectin expression by means of in vivo dividing T cells showed that L-selectin was expressed at high levels during the first five cell divisions (Fig. 3C), suggesting that the failure to detect expression of the α-chain of the IL-2 receptor during the first five cell divisions is not due to rapid proteolytic cleavage. To determine whether the T cells in the first five cell divisions are capable of expressing the α-chain of the IL-2 receptor, the T cells in the second cell division were classified, which did not express the α chain of the receptor of IL-2, and were stimulated in vitro with immobilized anti-CD3 and soluble anti-CD28 for three days. These classified T cells continued to divide by new in vitro stimulation, and all dividing T cells expressed to the α-chain of the IL-2 receptor. Clearly, the expression of the α-chain of the IL-2 receptor in vivo and in vitro is regulated differently.

Since the receptor for IL-15 also uses the γ and β chains of the IL-2 receptor as critical components of signaling (Tagaya et al., Immunity 4: 329-336, 1996), which are highly expressed during First five cell divisions, we wonder if the cells express an α-chain of the IL-15 receptor that makes them responsible for IL-15 during the initial cell divisions. The application of an IL-15-FLAG fusion protein as a primary coloring reagent (Chae et al., J. Immunol. 157: 2813-2819, 1996), demonstrated that the α-chain of the IL-15 receptor is clearly detectable , although at low levels, on dividing T cells regardless of the number of cell divisions (Fig. 4A). The α chain for the IL-2 receptor was not detected on all dividing T cells in vivo . Thus, the selective expression of the α chain for the IL-15 receptor, but not for the IL-2 receptor, together with the expression of the shared γ and β chains during the first five cell divisions, suggests that the initial cell division in vivo probably depends on IL-15 but not IL-2.

To analyze this hypothesis, the production of IL-2 in dividing T cells in vivo was evaluated. Intracellular staining of IL-2 revealed that IL-2 was highly expressed only by cells that have divided more than five times. Treatment of host mice with cytolytic anti-CD25 mAb saturation dose failed to inhibit the first five cell divisions (relative to control mice treated with Ab), and the dividing cells in the first and fifth divisions were remarkably similar in mice treated with anti-CD25 and in control mice. In addition, T cells were classified and cultured in vitro at a second cell division in vivo in the presence of IL-2 or IL-15, and cell proliferation was analyzed by assimilation of 3 H-TdR. IL-2, delivered in doses as high as 500 µ / ml in culture, failed to support T-cell proliferation. In contrast, IL-15 stimulated vigorous cell proliferation (Fig. 4B).

The expression pattern of IL-2 in vivo is closely associated with promoting the expression of the α and β chains of the IL-2 receptor, and with a markedly lower expression of the common γ chain (Fig. 5A ). This suggests that IL-2 regulates γ chain expression in vivo . To analyze this possibility, the expression of the γ chain was examined in the T cells of IL-2 deficient mice and in wild-type control mice. CD4 + T cells from IL-2 deficient mice expressed very high levels of γ chain on the cell surface compared to wild-type controls (Fig. 5B). Treatment of host mice with anti-CD25 inhibited the reduction of γ chain expression on dividing T cells in vivo , but this treatment had no effect on the expression of the IL-2 receptor β chain. (Fig. 5C).

The γ chain is a critical signaling element for all known T cell growth factors and γ chain signals are essential for cell survival, which is achieved at least in part through expression Sustained anti-apoptotic proteins of the Bcl-2 family ((Nakajima et al., J. Exp. Med. 185: 189-195, 1997). To determine whether the lowest γ chain expression after five cell divisions in vivo regulates clonal expression, CFSE-labeled cells were colored with PE-annexin V after in vivo analysis of host recovery and apoptotic cell death of dividing T cells. Precipitated cell death occurred after four cell divisions The undivided cells (division 0) had <10% positive cells with annexin V. After the sixth cell division, however, less than 40% of the cells were positive with annexin V. This type of cell death does not depend on Fas, since the T cells of Fas mutant MRL-1pr mice had a similar pattern of apoptotic cell death in vivo (Li et al., J. Immunol. 163: 2500-2507, 1999). The coloration for Bcl-2 expression showed that the average fluorescence intensity of the Bcl-2 coloration channel decreased markedly after four cell divisions (Fig. 5E). Thus, signaling events by reduction of γ chain expression may fail to support sustained expression of Bcl-2 and cells become susceptible to apoptotic cell death (Nakajima et al., J. Exp. Med 185: 189-195, 1997).

These results suggest that blocking IL-2 or IL-15 signaling will have different effects on the expansion of T cells in vivo . To explore this additional possibility, CFSE-labeled lymphocytes from IL-2 deficient mice (H-2b) were injected into irradiated DBA / 2 hosts (H-2d), cell division was analyzed in vivo three days later and compared. with that in wild-type control mice. T cells from IL-2 deficient mice continued to divide and expand in vivo . Approximately 30% of the CFSE-labeled cells entered the cell cycle, and most of the cells were divided more than five times, compared to control cells.

Treatment of host mice with an IL-15 / Fc mutant, which acts as a specific antagonist of the IL-15 receptor (Kim et al., J. Immunol. 161: 5742-5748, 1998), markedly reduced the frequency of proliferation of CFSE-labeled T cells, and an overwhelming majority of CFSE-labeled cells failed to enter the cell cycle in treated mice. In addition, treatment of host mice with blocking mAbs against the common γ chain, a shared signaling component of IL-2 and IL-15 receptors, also markedly inhibited the division of T cells in vivo . Thus, IL-2 and IL-15 regulate different aspects of T cell expression in vivo , and the administration of antagonists for these interleukins can suppress the immune response, as discussed above.

These results also demonstrate that the reduction of γ chain expression requires the activation of T cells and cell cycle progress as well as IL-2 signaling. Clearly, the reduction of γ chain expression in vivo is closely associated with the production of IL-2 with the high affinity expression of the IL-2 receptor. In the absence of IL-2, the γ chain is expressed at extremely high levels and the blocking of the IL-2 receptor inhibits the reduction of γ chain expression in vivo over the T-cell cycle. Thus , these studies provide new evidence that IL-2 and IL-15 regulate different aspects of primary T cell activation in vivo . Contrary to traditional beliefs and conclusions based on in vitro studies, IL-15 is a critical growth factor in the initiation of T cell division in vivo and the only role of IL-2 in vivo is to control the magnitude of clonal expansion by regulating the expression of the γ chain on the T cell cycle.

These results support applications clinics described above. Attempts to stimulate T cell response with exogenous IL-2 in Tumor immunity and in AIDS can promote the premature death of T cells and therapies to block IL-2 in induction of tolerance and autoimmunity can induce a unwanted expansion of T cells. In addition, the combined address and staged of IL-15 and IL-2 represents an important way to block the activation of T cells under cytopathic conditions that depend on cells T.

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IL-2 / Fc Lytic Lisan to the Supporting Cells to IL-2R and Bind to FcRI

The cells of a cell line were marked T (CTLL-2 cells; 10 6) with 100 mCi 51 Cr e incubated with a lytic form of IL-2 / Fc and a complement of low toxicity of rat (C '), a non-lytic form of IL-2 / Fc and C ', murine immunoglobulin and C' (a negative control) or C 'alone (a negative control of 0.5 µg / ml). The same cells were treated with a detergent (1% NP40) (a positive control). Cell lysis was measured by means of release of 51 Cr. The degree of lysis observed in the presence of the detergent represents a 100% lysis. The lysis was calculated specific after treatment as described above according to the formula: specific% of lysis = [(experimental cpm - background cpm) / (total cpm release - background cpm) x 100%]. The results shown in Fig. 6, support the conclusion that smooth cytolytic IL-2 / Fc to cells CTLL-2 that support IL-2R, but not The IL-2 / Fc does not lytic.

To assess the ability of the IL-2 / Fc lithic and non-lytic to bind to Fc receptors on CHO cells transfected with FcRI (FcRI of murine, FcRII, and IL-2R negative) were incubated previously FcRI transfectants with PBS, mIgG2a, IL-2 / Fc lytic, or IL-2 / Fc non-lytic. After washing, goat anti-mouse Fc was used fluorescent conjugate to color the cells for the FACS analysis. As shown in Fig. 7, at Cytolytic IL-2 / Fc may bind to FcRI, but not You can do it with lytic IL-2 / Fc.

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IL-2 / Fc Lítica is effective in preventing diabetes in an adoptive transfer model

Two different variants of IL-2 / Fc. The first contained a derivative Fc terminal of murine IgG2a that will mediate the CDC and the CDCC (IL-2 / Fc), and the second was a punctual portion Fc mutated that would not activate CDC or CDCC (IL-2 / Fc - / -). Although IL-2 / Fc media CDC in cells that support IL-2R (such as those of the murine CTLL-2 T cell line) and binds to FcRI, IL-2 / Fc - / - does not (Figs. 6 and 7). In addition, the lytic IL-2 / Fc molecule will prevent the development of diabetes in an adoptive NOD transfer model  but a non-lithic form of the molecule (IL-2 / Fc - / -) is ineffective (Fig. 13). In the model animal, the erythrocytes of the monodispersed cells of spleen by treatment with ACK Lysing Buffer (BioWhittaker Inc., Walkersville, MD). NOD male receptors irradiated from 8 to 12 weeks old (700 rad), who were not diabetic, were then injected with 2 x 10 7 splenic leukocytes from mice NOD female with acute diabetes (hyperglycemia <two weeks). Be analyzed weekly blood glucose levels (BGL), and diabetes was diagnosed when BGL was greater than 16.5 mmol / L on any individual measurement or greater than 13.8 mmol / L for 3 consecutive days. As shown in Fig. 13, the most animals remained free of diabetes even after the treatment was discontinued.

As noted above, the trials and Animal models presented here can be used to analyze different combinations of the agents of the invention for their therapeutic efficacy

Rapamycin inhibits the proliferation of T cells in an in vivo graft versus the host model but does not inhibit apoptosis of activated T cells

In an in vivo graft versus the host model (as described above), the fate of the host reactive T cells in the presence of IL-2 / Fc and rapamycin was analyzed. As shown in Fig. 8, rapamycin will inhibit the proliferation of CD4 + and CD8 + T cells as even in the presence of IL-2 / Fc, but will allow the expression of a functional IL-2R. As evidenced by staining with Annexin V, antigen-activated host reactive T cells will undergo apoptosis in the presence of IL-2 / Fc and rapamycin (Fig. 10).

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The compositions of the invention allow survival over a long period of skin grafting and islets of individuals of the same species

To avoid rejection of either the islets allogeneic transplants in NOD recipients with acute diabetes or the rejection of a layer of transplanted skin on NOD mice, it used a combination of agents that promote the death of T cells and the inhibition of T cell proliferation. As It is shown in Figs. 11 and 12, a combination of IL-2 / Fc lithic, mutIL-15 / Fc and rapamycin proved effective in preventing graft rejection (and more effective than other treatment regimens analyzed). The graft survival and graft function persisted during the entire observation period shown, and most of The grafts survived after discontinuation of therapy. This dramatic effect is believed to be due to the combination of agents used (agents that promote the death of T cells as well as inhibiting the proliferation of T cells).

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References cited in the description

This list of references cited by the Applicant is solely for the convenience of the reader. No way part of the European patent document. Although it has had great Be careful when collecting, you cannot exclude errors or omissions and the EPO rejects all responsibility in this regard.

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WO 9741232 A [0008]

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Claims (34)

1. A composition for use as a medicament that includes a first agent that is an interleukin antagonist 15 (IL-15), where the antagonist of IL-15 is a mutated polypeptide of IL-15 that has at least 65% identity with IL-15, and where the antagonist of IL-15 binds with and inhibits an activity of IL-15 or an IL-15 receptor, and a second agent that is an interleukin agonist 2 (IL-2), where the agonist is a polypeptide of IL-2, a mutated polypeptide of IL-2 or a receptor antibody anti-IL-2. 2. The therapeutic composition of the claim 1, wherein said antagonist of IL-15 is fused to an antigenic tag. 3. The therapeutic composition of the claim 1, wherein the first agent includes a polypeptide IL-15 mutant that binds to a subunit of an IL-15 receptor (IL-15R). 4. The therapeutic composition of the claim 3, wherein the subunit is a subunit of IL-15Rα. 5. The therapeutic composition of the claim 4, wherein the mutant polypeptide of IL-15 has a mutation at position 156 of the SEQ ID NO: 2. 6. The therapeutic composition of the claim 5, wherein the mutant polypeptide of IL-15 also has a mutation at position 149 of SEQ ID NO: 2. 7. The therapeutic composition of the claim 5, wherein the mutation at position 156 of SEQ ID NO: 2 is a substitution of aspartate for glutamine. 8. The therapeutic composition of the claim 6, wherein the mutation at position 149 of SEQ ID NO: 2 is a substitution of aspartate for glutamine. 9. The therapeutic composition of the claim 6, wherein the mutant polypeptide of IL-15 has an aspartate substitution for glutamine at positions 149 and 156 of SEQ ID NO: 2. 10. The therapeutic composition of the claim 3, wherein the first agent further comprises a fraction that leads to the elimination of the cells that support IL-15R. 11. The therapeutic composition of the claim 10, wherein the fraction is an Fc region of a IgG molecule. 12. The therapeutic composition of the claim 1, wherein the first agent comprises an antibody substantially anti-IL-15R pure. 13. The therapeutic composition of the claim 1, wherein the second agent comprises an antibody which specifically binds to an IL-2R. 14. The therapeutic composition of any of claims 1 to 13, further comprising rapamycin. 15. The use of an antagonist of IL-15, where the antagonist of IL-15 is a mutated polypeptide of IL-15 that has at least 65% identity with IL-15, and where the antagonist of IL-15 binds with and inhibits an activity of IL-15 or an IL-15 receptor, and a IL-2 agonist, where the agonist is a IL-2 polypeptide, a mutated polypeptide of IL-2 or a receptor antibody anti-IL-2 for the preparation of a therapeutic composition to suppress an immune response in a patient. 16. The use of claim 15, wherein said IL-15 antagonist is fused with a antigenic label 17. The use of claim 15, wherein the patient has an autoimmune disease or is at risk of develop an autoimmune disease 18. The use of claim 17, wherein the autoimmune disease is a rheumatic disease selected from group consisting of systemic lupus erythematosus, the syndrome of Sjögren, scleroderma, mixed tissue diseases connective, dermatomyositis, polymyositis, Reiter's syndrome and Behcet's disease 19. The use of claim 17, wherein the Autoimmune disease is rheumatoid arthritis. 20. The use of claim 17, wherein the Autoimmune disease is type I diabetes. 21. The use of claim 17, wherein the autoimmune disease is an autoimmune thyroid disease selected from the group consisting of Hashimoto's thyroiditis and Graves disease. 22. The use of claim 17, wherein the autoimmune disease is an autoimmune system disease central nervous selected from the group consisting of sclerosis multiple, myasthenia gravis, and encephalomyelitis. 23. The use of claim 17, wherein the autoimmune disease is a variety of pemphigus selected from the group consisting of vulgar pemphigus, vegetative pemphigus, pemphigus foliaceous, Senear-Usher syndrome, and pemphigus Brazilian. 24. The use of claim 17, wherein the Autoimmune disease is psoriasis. 25. The use of claim 17, wherein the Autoimmune disease is irritable bowel syndrome. 26. The use of claim 15, wherein the patient has acquired immunodeficiency syndrome (AIDS). 27. The use of claim 15, wherein the patient has received a transplant of an organ, tissue or cell Biological 28. The use of claim 15, wherein the patient has a graft versus host disease. 29. The use of an antagonist of IL-15, where the antagonist of IL-15 is a mutated polypeptide of IL-15 that has at least 65% identity with IL-15, and where the antagonist of IL-15 binds with and inhibits an activity of IL-15 or an IL-15 receptor, and a IL-2 agonist, where the agonist is a IL-2 polypeptide, a mutated polypeptide of IL-2 or a receptor antibody anti-IL-2 for the preparation of a therapeutic composition for the treatment of a disease malignant by IL-15R +. 30. The use of claim 29, wherein said IL-15 antagonist is fused to a tag antigenic 31. The use of claim 29, wherein the IL-15R + malignant disease is a leukemia, a lymphoma, or a colon cancer. 32. The composition of claim 1, in where the IL-2 agonist is a polypeptide of IL-2 33. The therapeutic composition of the claim 32, wherein the IL-2 polypeptide It also includes a fraction that leads to the elimination of cells that support IL-2R. 34. The therapeutic composition of the claim 33, wherein the fraction is an Fc region of a IgG molecule.